julilili42/papers huggingface dataset:
4-1BB Ligand Mediates a “Vicious Cycle” of Immune Evasion Upon Reciprocal Interaction of Chronic Lymphocytic Leukemia and NK Cells
2010-11-19

Abstract Abstract 2416 NK cells play an important role in tumor immunosurveillance. Their reactivity is governed by various immunoregulatory molecules, which influence both direct anti-tumor immunity and NK responses induced by therapeutic antibodies like Rituximab. Various members of the TNF/TNFR family modulate differentiation, proliferation, activation, and death of both tumor and immune effector cells including NK cells. Recently we reported that the TNFR family member 4-1BB/CD137 is expressed on human NK cells following activation. In contrast to the stimulatory role of its murine counterpart, we found that human 4-1BB impairs NK anti-tumor reactivity upon interaction with its ligand 4-1BBL expressed on blasts of a substantial proportion of acute myeloid leukemia patients (Blood 115: 3058-69; 2010). In addition, we found that expression of 4-1BBL is general feature of leukemic cells of chronic lymphocytic leukemia (CLL) patients causing impaired direct and Rituximab-induced NK cell reactivity (Blood 114: 279; 2009). Here we report that reverse signaling via 4-1BBL into CLL cells following interaction with 4-1BB, which is absent on NK cells of healthy donors, but expressed at substantial levels on NK cells of CLL patients, induced pronounced production of immunoregulatory cytokines like TNF, IL-6 and IL-8 by the CLL cells. Moreover, we found that sera of CLL patients contained elevated levels of these immunoregulatory cytokines as compared to healthy controls. When PBMC of healthy donors were exposed to supernatants of in vitro cultured CLL cells or sera from CLL patients, this resulted in pronounced 4-1BB expression on the NK cells. This effect could be prevented by addition of the TNF blocker Infliximab to patient sera. The 4-1BB expression induced by CLL sera resulted in impaired NK reactivity specifically against 4-1BBL-expressing targets as revealed by functional analyses with 4-1BBL transfectants and the respective mock-controls. Moreover, the induced 4-1BB expression also impaired NK cell reactivity against primary CLL cells constitutively expressing 4-1BBL, thus closing a cycle of immune evasion. Taken together, our data demonstrate that 4-1BBL enables CLL cells to evade NK anti-tumor reactivity, and disruption of the “vicious 4-1BB-4-1BBL cycle” in CLL - NK interaction e.g. by TNF- or 4-1BB-blockade may serve well to enhance NK reactivity in therapeutic strategies like antibody treatment or allogenic stem cell transplantation in CLL, which rely on sufficient NK cell function. Disclosures: No relevant conflicts of interest to declare.
Incidence and Outcome of Auto/Alloimmune Hepatitis with Hepatic Graft-Versus-Host Disease After Hematopoietic Stem Cell Transplantation.
2010-11-19

Abstract Abstract 4534 Auto/alloimmune hepatitis (AIH) is an inflammatory liver disease characterized histological by a dense mononuclear cell infiltrate in the portal tract and serological by the presence of non-organ and liver-specific antibodies, high transaminases and increased levels of IgG. The relation between allogeneic hematopoietic stem cell transplantation (HSCT) and auto/alloimmune disease is complex. To examine this association, we retrospectively studied 1,636 allogeneic patients (median age 43, range 18–73 years) between May 1996 and December 2008. Among these patients, 311 (19%) developed hepatic graft-versus-host disease (GvHD) (162 pts had a hepatic GvHD of grade &gt; II). We followed 25 patients (11 male, 14 female) in whom GvHD of the liver presented with marked elevation of serum aminotransferases, clinically resembling acute hepatitis and auto/antibodies characteristics for AIH. The median age at transplant was 35 (range, 18–54) years. Onset of liver dysfunction was at 286 days (range, 55–2766) after HSCT. Median peak serum was 312 (range 105–1750) U/L for alanine aminotransferase, 629 (133-2410) U/L for gamma-glutamyl transferase and 1.74 (0.5-23.4) mg/dl for bilirubin. The autoantibody profiles of AIH were 60% for anti-nuclear antibody, 44% for antibodies to liver-kidney microsomes, 24% for antibodies to smooth-muscle antigens, 28% for anti-mitochondrial antibody, 16% for antibodies to actin, 8% for antibodies to nucleoli, and 4% for other autoantibodies. AIH had a higher prevalence in younger and in female patients. AIH occurred in 92% in patients, who were transplanted with G-CSF mobilized and peripherally collected stem cells (PSC), but in only 8% in patients with bone marrow (BM) source (p&lt;0.02), comparing all transplanted patients (1326 PSC, 310 BM). Stem cell grafts from matched sibling donor or matched unrelated donor were similar in the two groups. Acute GvHD of grade&gt; II occurred more frequently in the groups with AIH (15/25 vs. 649/1636, p&lt;0.002), and chronic GvHD (11 limited, 14 extensive) was ascertained in all AIH patients vs. 49.8% in all transplanted patients (p&lt;0.0001). Three patients with AIH died from pulmonary bleeding, chronic GvHD, and relapse, whereas 22 patients with AIH are still alive (88%) at a median survival time of 2570 days. In conclusion, our evaluation confirms a strong association between G-CSF mobilized PSC, chronic GvHD and the development of AIH. Disclosures: No relevant conflicts of interest to declare.
Cysteinyl Leukotrienes Signal Via Overexpressed CysLT1, but Not CysLT2 Receptor In Chronic Lymphocytic Leukemia
2010-11-19

Abstract Abstract 3612 The G Protein-coupled receptors (GPCRs) CysLT1 and CysLT2 are both expressed in peripheral blood mononuclear cells (PBMNC). Their ligands are inflammatory mediators of the cysteinyl leukotriene (cysLT) family and contribute, together with ligands of other GPCRs such as chemokines, to migration and proliferation of a variety of cell types. In the present study, we demonstrate by real-time RT-PCR that CysLT1 mRNA is overexpressed in B-CLL cells compared to both normal PBMNC and normal CD19+ B cells, whereas only low levels of CysLT2 mRNA are present. GPCR-typical cellular responses such as intracellular calcium fluxes and actin polymerization, which were induced by the cysLT LTD4 in CLL cells in a dose-dependent manner, were suppressed by the CysLT1 antagonist MK571, the prototype of the lukast family of drugs used in asthma treatment (e.g., montelukast). However, MK571 and other lukasts also block the leukotriene transporter MRP. The fact that the CysLT1 antagonist LY171883, which has no effect on MRP, also abrogated the responses of CLL cells indicates that the effects of cysLTs are mediated solely by CysLT1. Moreover, also chemotaxis was induced by the cysLT LTD4 in B-CLL cells (optimum at low nanomolar concentrations) and could be blocked by MK571. We further observed inhibiting effects of MK571 on cysLT-induced Erk/MAP kinase phosphorylation in B-CLL cells, which suggests an involvement of CysLT1 in cell proliferation. Indeed, MK571 significantly induced apoptosis of CLL cells in vitro and reduced survival of cultured B-CLL cells. However, the concentrations for a significant reduction of cell viability were higher (1-10 μM) than the concentration required for efficient CysLT1 receptor inhibition (0.1 μM). Thus, the influence of MK571 on B-CLL cell survival may mainly be due to direct effects on apoptosis occuring at the higher dose level and/or blocking of the leukotriene transporter MRP. Accordingly, LY171883 and the combined CysLT1/2 antagonist Bay-u9773 did not induce CLL apoptosis. Our results suggest that CysLT1 is overexpressed in B-CLL cells and involved in cell trafficking, similar to the chemokine receptor CXCR4. Considering the redundancy among GPCRs and the limited effect of MK571 on cell survival, CysLT1 for its own represents a less attractive therapeutic target using the currently available CysLT1 antagonists of the lukast family. However, combined inhibition of several GPCRs highly expressed in CLL cells including cysLT1 and CXCR4 may constitute a promising therapeutic approach and should be further evaluated. Disclosures: No relevant conflicts of interest to declare.
Incipient Release of Leukocytes After Allogeneic Hematopoietic Cell Transplantation Is Indicated by Increasing Serum Levels of Uric Acid Before Detection of First Leukocytes
2010-11-19

Abstract Abstract 4701 Uric acid serves as an endogenous danger signal to the immune system upon the release from dying cells activating both innate and adaptive immunity. Multiple cytokines and danger signals are released into the blood modulating cellular and humoral immune responses during haematopoietic cell transplantation (HCT). Here, we investigated serum levels of uric acid during haematopoietic regeneration after allogeneic HCT to evaluate whether uric acid can be used as marker for hematopoietic engraftment indicating immunologic activity below the detection limit of differential blood counts (50/μ l). Differential blood counts and serum levels of uric acid, creatinine and urea were retrospectively analyzed in 47 consecutive patients (22 men and 25 women, median age 48 years, range 19 to 73 years) during 50 allogeneic HCT (three patients received a second transplantation due to recurrent disease). Serum levels of uric acid, creatinine and peripheral blood counts were determined daily. All patients received allopurinol for 5 days from the beginning of the conditioning which was discontinued afterwards. The mean nadir in uric acid levels was observed 13.4 days after the last dose of allopurinol (range 2 to 32 days) and 9.1 days after transplantation (range 2 to 21 days) irrespective of the conditioning regimen used. In all patients a significant decrease of uric acid serum levels after the transplantation was observed (Student's t-test, p &lt; 0.001). A re-increase of uric acid was observed 1 to 10 days (mean 4.1 days, SD 2.4) before detection of white blood cells (leukocytes &gt; 50/μ L) in 35 out of 50 transplantations (70%, group 1). In three cases (6%), the increase of uric acid levels was observed concomitantly with increasing leukocytes (group 2). In the remaining 12 cases, this elevation was only observed later (1 to 6 days after re-increase of leukocytes, mean 2.8 days, SD 1.5, group 3). The difference between group 1 and 3 was statistically significant (Student's t-test, p &lt; 0.001). No correlation with patient age, conditioning regimen, HLA compatibility or additional medications could be observed. Therefore, a re-increase of uric acid can be indicative for incipient haematopoietic engraftment after allogeneic HCT and may serve as indicator for consumption of the ealiest differentiated cells in peripheral blood even before leukocytes can be detected. Disclosures: No relevant conflicts of interest to declare.
The Immune-Inhibitory Receptor Osteoactivin Is up-Regulated In Monocyte-Derived Dendritic Cells Upon Exposure to Tyrosine Kinase Inhibitors
2010-11-19

Abstract Abstract 1733 Targeted therapies with tyrosine kinase inhibitors (TKI) have significantly improved the treatment of cancer patients. Ex vivo generated dendritic cells (DC) are commonly used in immunotherapeutic strategies due to their unique ability to initiate adaptive immune responses, and multiple approaches presently aim to combine targeted therapies with immunotherapy. However, as many kinases targeted by TKI are, besides governing tumor cell growth, also involved in the activation of DC, TKI therapy may cause immunoinhibitory side effects. Osteoactivin (GPNMB, DC-HIL) is a type I transmembrane glycoprotein that is detected abundantly in DC but not in monocytes. Its expression on antigen-presenting cells can inhibit T cell activation by binding syndecan-4 (SD-4) on T cells. Here we investigated the effect of the BCR/ABL TKI imatinib, dasatinib and nilotinib, which are approved for the treatment of CML, on the expression of osteoactivin and DC functions. DC were generated from blood monocytes by plastic adherence and exposure to GM-CSF and IL-4. Imatinib, nilotinib or dasatinib were added to the culture medium every second day starting from the first day of culture. In some experiments, toll-like receptor (TLR) ligands (L) (LPS (TLR4L), pam3Cys (TLR2L), poly I:C (TLR3L) or R848 (TLR7/8L) were added on day 6 of culture for maturation of DC. We found that DC generated in the presence of therapeutic concentrations of all three TKI displayed an altered phenotype. Imatinib caused significantly reduced expression of the typical DC markers CD1a, CD83 and the co-stimulatory molecule CD86. Nilotinib reduced the expression of CD1a, CD83, CD86 and the DC-specific C-type lectin receptor DC-SIGN (CD209). Dasatinib impaired expression of CD1a, CD83, CD86, CD80 and DC-SIGN. Most notably, we observed excessive up-regulation of osteoactivin on DC upon treatment with all three TKI. Interestingly, incubation with the immunosuppressive and anti-inflammatory cytokine IL-10 also resulted in osteoactivin over-expression. In line with osteoactivin up-regulation, exposure to TKI resulted in reduced stimulatory capacity of DC in MLR with allogenic T cells that could be restored by addition of blocking anti-osteoactivin antibody. In summary, our data demonstrate that up-regulation of osteoactivin is critically involved in the inhibition of DC function upon TKI exposure. These findings are of great importance for future combinatory approaches using TKI and DC-based immunotherapy and indicate that inhibition of osteoactivin expression or function may serve as a novel strategy to enhance the efficacy of immunotherapeutic interventions in cancer patients. Disclosures: No relevant conflicts of interest to declare.
The Heatshockprotein-90 Inhibitor IPI-504 Potently Overcomes Tyrosinekinase Inhibitor-Induced Activation of Cell Protection Mechanisms and Degrades HSP-Client Proteins KIT and FLT3 In Acute Leukemia Models
2010-11-19

Abstract Abstract 3982 Activating mutations of FLT3 and KIT class III receptor tyrosine kinases (TK) are associated with acute leukemias and systemic mastocytosis. Despite the encouraging therapeutic potential of TK inhibitors (TKI), monotherapy often is not sufficient to obtain lasting responses. Here we show that TKI therapy may lead to cellular protection mechanisms including stabilization/repair of DNA, as well as protection of the function of heat shock proteins. We further demonstrate that degradation of heat shock protein 90 (HSP90) client proteins, FLT3 and KIT, by the HSP90 inhibitor IPI-504, leads to antitumor effects in acute leukemia and mastocytosis models; including additive to synergistic antiproliferative and proapoptotic activity after TKI treatment. To explore the immediate downstream signaling effects of mutant FLT3 and KIT inhibition in acute leukemia and mastocytosis models, we performed unbiased phoshoproteomic analyses of cells before and after TKI treatment. Phosphopeptides that displayed a significant difference in phosphorylation before and after FLT3/KIT inhibition with Imatinib or Sunitinib at IC90 were immunoaffinity purified, and identified by tandem mass spectrometry. Degradation of FLT3 and KIT after HSP90 inhibition was studied by immunoblotting. Antiproliferative and proapoptotic effects of the HSP90 inhibitor IPI-504 alone, or in combination with TKI treatment, were studied in different leukemia and mastocytosis cell models, and in an isogenic BaF/3 model harboring different clinically relevant KIT and FLT3 mutations. Phoshoproteomics revealed a significant upregulation in phosphorylation of DNA and protein function stabilizing proteins, including HSP members in mutant KIT and FLT3 cell lines. Both FLT3 and KIT turned out to be client proteins of HSP90, and IPI-504 was able to degrade FLT3 and KIT protein, resulting in a strong potentiation of the antiproliferative and proapoptotic effects achieved by TK inhibitors alone. Of interest, IPI-504 displayed antitumor activity even as monotherapy in some leukemia and mastocytosis cell line models. Our data suggest that TK inhibition of FLT3 or KIT causes initiation of cell protective mechanisms, including activation of HSPs, which can therapeutically be targeted by HSP90 inhibitors such as IPI-504. These results provide a rationale to combine TKI with HSP inhibitors to optimize TKI therapy in patients with hematopoietic malignancies. Disclosures: Normant: Infinity pharmaceuticals: Employment.
Fc-Engineered RANK-Fc Fusion Proteins for Neutralization of Soluble RANKL and Induction of Antibody-Dependent Cellular Cytotoxicity (ADCC) Against Multiple Myeloma
2010-11-19

Abstract Abstract 3039 Bone resorption is commonly associated with aging, but also with certain cancers. Recent studies identified Receptor Activator of NF-κB (RANK) ligand (RANKL) and its receptors RANK and osteoprotegerin as key regulators of bone remodelling. Multiple myeloma (MM) disrupts the balance within this molecule system towards osteoclastogenesis and bone destruction. Neutralization of RANKL by the monoclonal antibody Denosumab (AMG162) is presently being evaluated for treatment of both non-malignant and malignant osteolysis. We found, in line with previously published data, that primary MM cells (9 of 10) express substantial levels of RANKL at the cell surface and that MM cells directly release RANKL in soluble form (sRANKL). Next we evaluated the possibility to combine neutralization of sRANKL with targeting of MM cells for antibody-dependent cellular cytotoxicity (ADCC) of NK cells utilizing RANK-Ig fusion proteins with modified Fc portions. Compared to wildtype RANK-Fc, our mutants (S239D/I332E and E233P/L234V/L235A/DG236/A327G/A330S) displayed highly enhanced (RANK-Fc-ADCC) and abrogated (RANK-Fc-KO) affinity, respectively, to the NK cell FcγRIIIa, but comparable capacity to neutralize RANKL in binding competition and osteoclast formation assays. Analyses with RANKL transfectants and RANKL-negative controls confirmed the high and lacking potential of the RANK-Fc-ADCC and the RANK-Fc-KO to induce NK ADCC, respectively, and ascertained that the RANK-Fc-ADCC specifically induced NK cell lysis of RANKL-expressing but not RANKL-negative target cells. Most notably, in cultures of NK cells with RANKL-expressing primary MM cells RANK-Fc-ADCC potently enhanced NK cell degranulation, cytokine release and MM cells lysis due to enhanced NK reactivity. Thus, our Fc-engineered RANK-Fc-ADCC fusion protein may both neutralize detrimental effects of sRANKL and enhance NK anti-tumor reactivity by targeting RANKL-expressing malignant cells thereby constituting an attractive immunotherapeutic means for treatment of MM. Disclosures: No relevant conflicts of interest to declare.
His or mine? The time course of self–other discrimination in emotion processing
2010-11-20

This electroencephalography (EEG) study investigated at which temporal processing stages self-other discrimination in emotion processing occurs. EEG was recorded in 23 healthy participants during silent reading of unpleasant, pleasant, and neutral pronoun-noun and article--noun expressions that were related to the participants themselves, related to an unknown third person, or had no self-other reference at all. Self- and other-related pronoun--noun pairs elicited larger cortical negativity relative to the processing of article--noun pairs at left posterior electrodes as early as 200 ms after stimulus onset. In the same time windows (from 200 ms to 300 ms and 300 ms to 400 ms) the emotionality of the words enhanced event-related brain potential (ERP) amplitudes at parieto-occipital electrodes. From 350 ms onwards, processing of self-related unpleasant words elicited larger frontal negativity compared to unpleasant words that were related to the other or that had no reference at all. In addition, processing of pleasant words vs. neutral or unpleasant words elicited larger positive amplitudes over parietal electrodes from 450 ms after stimulus onset, in particular when words were self-related. Our findings demonstrate that for verbal emotional stimuli, self--other discrimination first occurs at higher-order, cortical processing stages. This is consistent with the view that categorization of information according to certain stimulus aspects (self--other reference, emotionality) occurs before its meaning is integrated.
TRPV Channel Expression in Human Skin and Possible Role in Thermally Induced Cell Death
2010-11-20

Cell death via necrosis and apoptosis is a hallmark of deep dermal to full-thickness cutaneous burn injuries. Keratinocytes might act as thermosensory cells that transmit information regarding ambient temperature via heat-gated transient receptor potential vanilloid (TRPV) ion channels. The aim of this study was to investigate the distribution of TRPV1, 2, 3, and 4 in uninjured and thermally burned skin. The authors investigated warmth-evoked currents in keratinocytes and cell kinetics of thermally injured keratinocytes in culture with agonists and antagonists of TRPV channels. Specimens of uninjured normal skin and discarded tissue of thermally injured skin were stained for TRPV1, 2, 3, and 4. Cultured primary human keratinocytes were heated for 5 minutes at the following temperatures: 37°C (control), 42°C, and 60°C and thereafter cultured for 24 or 48 hours at 37°C. Thermally stressed cells were treated with TRPV antagonists capsazepine or ruthenium red, and cell viability capacity was determined. TRPV1, TRPV2, TRPV3, and TRPV4 immunoreactivity was differentially identified on basal and suprabasal keratinocytes of healthy human skin. Patch clamp analysis showed a functional response of human keratinocytes at temperatures >40°C. Cell death of keratinocytes after heating at 42°C was reduced by 15 and 5% with ruthenium red and by 20 and 30% by capsazepine at 24 and 48 hours, respectively. Cell death after treatment at 60°C was significantly reduced at 24 hours with capsazepine (22%) or ruthenium red (18%) but only minimally affected after 48 hours postinjury. Interaction with TRPV channels on keratinocytes may offer a new strategy to counteract cell death after thermal injury.
Underestimated Risk of Bleeding after Male Transobturator Sling Procedure Caused by Early Re-Uptake of Anticoagulation
2010-11-20

The management of postoperative male stress urinary incontinence (SUI) is changing rapidly as greater emphasis on minimizing invasiveness and reducing complications leads to technical innovations. Various studies have reported comparable continence but lower complication rates for male urethral slings than those observed with artificial urinary sphincters. For this reason, the transobturator sling procedure has been considered a safe and effective treatment option in male SUI patients. In this case study we report severe bleeding with a large hematoma that required transfusion after implantation of an AdVance® male transobturator polypropylene sling in an early re-anticoagulated patient.