IFN-gamma Expression Research Articles

The roles of Galectin-3 in autoimmunity and tumor progression

Galectin-3, a unique chimera-type member of the β-galactoside-binding soluble lectin family, is widely expressed in numerous cells. Here, we discuss the role of Galectin-3 in T-cell-mediated inflammatory (auto) immunity and tumor rejection by using Galectin-3-deficient mice and four disease models of human pathology: experimental autoimmune encephalomyelitis (EAE), Con-A-induced hepatitis, multiple low-dose streptozotocin-induced diabetes (MLD-STZ diabetes) and metastatic melanoma. We present evidence which suggest that Galectin-3 plays an important pro-inflammatory role in Con-A-induced hepatitis by promoting the activation of T lymphocytes, NKT cells and DCs, cytokine secretion, prevention of M2 macrophage polarization and apoptosis of mononuclear cells, and it leads to severe liver injury. In addition, experiments in Galectin-3-"knock-out" mice indicate that Galectin-3 is also involved in immune-mediated β-cell damage and is required for diabetogenesis in MLD-STZ model by promoting the expression of IFN-gamma, TNF-alpha, IL-17 and iNOS in immune and accessory effector cells. Next, our data demonstrated that Galectin-3 plays an important disease-exacerbating role in EAE through its multifunctional roles in preventing cell apoptosis and increasing IL-17 and IFN-gamma synthesis, but decreasing IL-10 production. Finally, based on our findings, we postulated that expression of Galectin-3 in the host may also facilitate melanoma metastasis by affecting tumor cell adhesion and modulating anti-melanoma immune response, in particular innate antitumor immunity. Taken together, we discuss the evidence of pro-inflammatory and antitumor activities of Galectin-3 and suggest that Galectin-3 may be an important therapeutic target.

Relationship of Cytokine and Regulatory Gene Expression to the Outcomes of Milk and Egg Allergy in an Atopic Cohort (COFAR2)

S A T U R D A Y 94 Relationship of Cytokine and Regulatory Gene Expression to the Outcomes of Milk and Egg Allergy in an Atopic Cohort (COFAR2) S. H. Sicherer, D. Stablein, R. A. Wood, A. W. Burks, A. H. Liu, S. M. Jones, D. M. Fleischer, L. Mayer, R. Lindblad, A. Grishin, H. A. SampsonConsortium of Food Allergy Research. Mt. Sinai School of Medicine, New York, NY, EMMES Corp., Rockville, MD, Johns Hopkins University School of Medicine, Baltimore, MD, Duke, Durham, NC, National Jewish Health, Denver, CO, University of Arkansas for Medical Sciences, Little Rock, AR. RATIONALE: To determine lymphocyte activation biomarkers associated with milk/egg allergy over time. METHODS: Children aged 3-15 months with likely milk/egg allergy and no diagnosed peanut allergywere followed at baseline, 6, 12 and 24months and categorized as egg/milk allergic or tolerant by predefined criteria. Mononuclear cell allergen stimulation screening for CD25, CISH, FOXP3, GATA3, IL-10, IL-4, IFN-gamma and TBET expression using casein and egg white as stimulants were performed at each of the time points. Repeated measures analyses with generalized linear modeling were constructed to determine if gene expression was predictive of the allergy state (allergic or tolerant). RESULTS: The number of children with a confirmed/convincing milk allergy and number tolerant in parentheses among those tested at baseline (n5492), 6 (n5432), 12 (n5420) and 24(n5367) months were: 237(105), 197(137), 197(163) and 145(190), respectively. The corresponding results for egg were: 139(23), 132(35), 129(55) and 99 (117), respectively. The significant (p < 0.05) predictors of allergy based upon current gene expression values were: increased IL4 expression (milk) and decreased GATA3 (egg). Considering change in activation from baseline, risk was associated with a decrease in TBET (milk) and increased IL4 (egg). Tolerance to egg was associated with current decreased CISH, and decreased TBET compared to baseline. However, the predictive capacity of the findingswasminimal, with only a few percentage points of risk being predicted by large changes in expression. CONCLUSIONS: Allergen-stimulated lymphocyte gene expression failed to significantly reflect allergy states over time.

DC-SIGN modulates DC maturation and function in rat renal tubulointerstitial lesions

The role of DC-SIGN in tubulointerstitial lesions (TILs) and the effect of anti-P-selectin lectin-EGF domain monoclonal antibody (PsL-EGFmAb) were investigated in rat nephrotoxic nephritis (NTN). On Day 4, immature DC-SIGN+DCs infiltrated into renal tubulointerstitium and matured by Day 14, showing increased migratory capacity and ability to induce T cell proliferation. The distribution of DC-SIGN+ DC significantly correlated with crescent formation, TIL severity, and changes in renal function. RANTES and TNF-alpha mRNA were continuously up-regulated from Day 4, while IL-10 mRNA was down-regulated after a marked increase on Day 4. Expression of IFN-gamma and IL-4 mRNA increased on Day 14 due to DC maturation. PsL-EGFmAb suppressed DC maturation, migration and ability to activate T cells. It also down-regulated TNF-alpha and up-regulated IL-10, resulting in a Th1/Th2 bias. The number of crescents decreased and TILs and renal function improved. These results suggest that DC-SIGN mediates DC tubulointerstitial infiltration and is an important regulator of local immune reactions and TILs. PsL-EGFmAb inhibited DC migration, maturation and function by targeting DC-SIGN, and may therefore be a potential treatment for NTN.

Cytokine responses to Mycobacterium leprae unique proteins differentiate between Mycobacterium leprae infected and naïve armadillos

New diagnostic tools for early detection of leprosy are necessary to help reduce its transmission and severity. M. leprae unique proteins have been used to assess differences in human T-cell responses in leprosy patients, household contacts and endemic controls. In this study, we examined the response of M. leprae-infected armadillos to a variety of M. leprae recombinant antigen candidates currently being examined for diagnostic efficacy in humans. Among recently M. leprae infected armadillos, IFN-gamma expression was enhanced after stimulation of PBMC with all M. leprae recombinant proteins except for ML2283 (mean: 2.65 Relative Quantification (RQ)). The group mean stimulation index for M. leprae proteins ML0009, ML1601, ML2478 and ML2531 averaged 35.2 RQ and was significantly higher (P < 0.05) than that measured among the non-infected, naive group (mean 6.2 RQ). Although ML0840 tended to enhance IFN-gamma levels, the mean IFN-gamma transcript levels of the currently experimentally inoculated group (20.1 RQ) was not significantly different statistically (P = 0.10) from the mean of the naive group (7.5 RQ). Also no statistically significant differences were observed in IFN-gamma transcript levels between the resistant and currently experimentally inoculated group (P > 0.05) or between the resistant and the naive group (P > 0.05) after stimulation of PBMCs with all M. leprae recombinant proteins. Only low levels of TNF-alpha were observed across all groups after in vitro stimulation with all the antigens examined. These data suggest that armadillos can be used effectively to help identify M. leprae specific proteins that may be applied for monitoring T-cell responses in M. leprae infected hosts as their disease progresses as well as for the early diagnosis of leprosy.

Abstract 9434: A Specific IKK Inhibitor Suppresses Experimental Autoimmune Myocarditis in Rats

Introduction: Nuclear factor-kappa B (NF-κB), which is regulated by the inhibitor NF-κB kinase (IKK), plays a critical role in the development of myocarditis. However, little is known about the role of IKK in its pathophysiology. Hypothesis: We assessed the hypothesis that IKK inhibition could attenuate cardiac inflammation in the progression of myocarditis. Methods: Lewis rats were immunized with porcine cardiac myosin to induce experimental autoimmune myocarditis (EAM). We administered the IKK inhibitor (IMD-0354, 15mg/kg/day, n=6) to EAM rats daily; the drug-free vehicle was used as untreated EAM control (n=7). We analyzed the therapeutic effects of IKK inhibition against EAM using echocardiography and pathological examination. Cytokine expression in the hearts and cell proliferation by myosin restimulation were also analyzed. Results: Echocardiogram revealed that the IKK inhibitor improved left ventricular fractional shortening (52.7±4.5%) in comparison to the untreated group (37.3±3.1%, p&lt;0.05). Pathologically, severe myocardial cell infiltration was observed in the untreated EAM group (affected area per whole heart, 55.8±6.1%), while the IKK inhibitor treated group showed to suppress these changes (26.3±4.2%, p&lt;0.05). In the IKK inhibitor treated group, mRNA expression of IFN-gamma (3.9 ±0.7 fold vs. untreated group, p&lt;0.05) and MCP-1 (17.1±0.8 fold vs. untreated group, p&lt;0.05) was significantly suppressed in comparison to the untreated EAM group (IFN-gamma: 14.5±2.7 fold, MCP-1: 51.4±3.9 fold). Moreover, the IKK inhibitor suppressed production of IL-2 and T cell proliferation in a dose dependent manner in vitro. Conclusion: In conclusion, the IKK inhibitor ameliorated EAM by suppressing cytokine production and attenuating inflammatory cell infiltration. IKK regulation is a promising treatment of clinical myocarditis.

Low Blood Levels of Th17 Cells Are Seen in Patients with aGvHD and Associate with Rather Poor Survival Post HSCT,

Abstract 4079Alloreactivity strongly influences the course post HSCT being a primary force in pathomechanism of aGvHD, shapes the immunological reconstitution post-transplant and mounts graft vs leukemia effect. Therefore the understanding of the interplay between the main subsets of CD4 positive cells may help in understanding the mechanism which facilitates the activity of the immune system post HSCT. For that we investigated the presence of cells with a potential to generate IL-17, IFN-gamma and FoxP3 lymphocytes appearing in blood to post HSCT.The cytoplasmic expressions of IL-17A, FoxP3 and IFN-gamma were studied in stimulated PBMC (brefeldin A, Ionomycin and PMA) of alloHSCT patients (63 patients, median age: 43 yrs, range: 5.5 –60 yrs), 12 patients manifested aGvHD at the time of hematological recovery and in 17 patients aGvHD was clinically apparent after hematological reconstitution (from 17 to 93, median 31). The cells were labeled for CD4 (Becton Dickinson, San Jose, CA, USA) and intracellular IL-17A (e-biosciences, San Diego, CA, USA), FoxP3 and IFN-gamma (Becton Dickinson, San Jose, CA, USA) detection. All studied cases were genotyped for NOD2/CARD15 mutations: 104C-T (SNP8, Arg702W; rs.2066844), 2722G-C (SNP12, Gly908Arg; rs.2066845), and 3020insC (SNP13, Leu1007fsinsC; rs.2066847) with the use of RFLP-PCR technique.Patients post HSCT studied at the beginning of hematological recovery (1st observation point) had lower blood levels of IL-17 producing lymphocytes in CD4+ cells (Th17) independently whether they developed aGvHD at the hematological recovery or (later time) post-transplant as compared to those lacking aGvHD (median 0.090% (0.195%) vs 0.425%, p=0.019 (p=0.126)).Primarily low Th17 blood level in aGvHD cases normalized (increased) along the observation time, when a positive response to the therapy was noticed, being in one week intervals in the levels as follow (1st vs 2nd (3rd) time point: median 0.090% vs 0.355% (0.355%), p=0.049 (p=0.061)). Of note, patients lacking aGvHD at any time post HSCT had similar blood levels of Th-17 cells during 30 days post-transplant observation time.The first quartile of Th17 blood values of the whole group was 0.1% and this value was chosen as a cut-off point dividing the whole group into patients with low and higher blood Th17 levels. It became apparent that survival of patients having low Th17 blood levels at the beginning of hematological recovery, irrespective of the presence of absence of aGvHD, had poorer survival (2 yrs survival 30% vs 61%, p=0.045). When fatal cases were analyzed patients with low Th17 blood levels died rather due to aGvHD and those with higher levels due to infectious complications or relapse (7/10 vs 4/16, p=0.043).FoxP3+ cells and IFN-gamma producing lymphocytes were determined in blood of HSCT patients at the same time as it was done for Th17 cells. It was shown that the blood levels of FoxP3+ cells were well correlated with the levels of IFN-gamma producing cells (r=0.405, p=0.003) but not to the similar extent with Th17 cells (r=0.253, p=0.047).Th17 plays an important role in the mucous membrane barrier against microbial infections. Mutations in NOD2/CARD15 gene are described as a risk factor of aGvHD. We found that mutations in the NOD2/CARD15 gene is not more frequent in aGvHD cases but influences the level of Th17 in blood in such a way that patients with mutations had lower blood values of Th17 at the hematological recovery in the aGvHD group (median 0.080% vs 0.210%, p=0.051) as well as in the whole alloHSCT patient group (median 0.080% vs 0.310%, p=0.027).Conclusions:1.Th17 lymphocytes being lower at the beginning of aGvHD manifestation likely to marginalize at the sites of inflammation.2.Low levels of Th17 were associated with rather poor survival of HSCT pts with aGvHD as a primary cause of death.3.NOD2/CARD15 mutations associate with lower values of Th17 in blood.Supported by the grants N N402 430039 and N R13 0082 06 from the Polish Ministry of Science & Higher Education. Disclosures:No relevant conflicts of interest to declare.

Influence of four parts of forming of Yiqi Qingwen Jidu Heji(YQQWJDHJ) to lung inflammatory cytokines of model rats infected with influenza virus FM1

To observe the influence of the 4 parts of forming of Yiqi Qingwen Jidu Heji(YQQWJDHJ) to lung inflammatory cytokines of the model rats infected with influenza virus dynamically, and to discuss the mechanism of 4 parts of forming to anti-influenza immune injury and restoration. At the different stages of infection with the model rats infected by FM1 influenza, expression in lung of TNF-alpha, IL-6, IL-1, IL-10 and IFN-gamma was detected after the intervention of 4 parts of forming using ELISA method. The expression of TNF-alpha, IL-6, IL-1 and IFN-gamma of model rats infected by FM1 were higher than the control group, the expression of IL-10 did not change. The expression of TNF-alpha was significantly reduced in 3 to 5 days after infection. By the method of relieving superficies with acrid-cold, clearing away heat and poison and replenishing Qi, the lung expression of IFN-gamma was significantly increased in the stage after infection. The method of relieving superficies with acrid-warm significantly reduced lung expression of IL-6 after infection in 1 to 3 days and on the 7th day, decreased the expression of IL-1 in 3 to 7 days, increased IFN-gamma expression on the 3rd day and the 7th day, and significantly increased the expression of IL-10 on the 1st day and in 5 to 7 days. The method of relieving superficies with acrid-cold reduced the expresssion of IL-6 after infection, and significantly increased the expression of IL-10. It could increase the expression of IL-1 after infection on the 3rd day, but reduced IL-1 expression after infection 7 days. The method of clearing away heat and poison reduced lung IL- 6 expression after infection in 3 to 7 days significantly, decreased the expression of IL-1 in 5 to 7 days, also increased the lung expression of IL-10 in 1 to 5 days significantly. The method of replenishing Qi significantly reduced the expression of IL-6 after infection on the 1st day and in 5 to 7 days, decreased the expression of IL-1 in 3 to 7 days, also significantly increased the lung IL-10 on the 5th day after infection. The method of clearing away heat and poison and replenishing Qi could be against the lung immune inflammatory damage and repair damage. The method of relieving superficies with acrid-warm demonstrated some immunity against lung injury on the 3rd day after infection and the method of relieving superficies with acrid-cold demonstrated some immunity against lung injury on the 5th days after infection.

Chronic alcohol consumption induces cardiac remodeling in mice from Th1 or Th2 background

Aims The effects of T helper (Th) cells on alcoholic cardiomyopathy have not been extensively investigated. Strain of mice with Th1 or Th2 immunological background were utilized in this study in order to explore the role of Th1/Th2 in chronic alcohol-induced cardiac fibrosis. Methods and results C57BL/6 WT or Balb/c mice were treated with alcohol for 90 days. Then cardiac structure and function were analyzed via echocardiography. Spleen CD4 + CD25 + Foxp3+ Tregs were determined by flow cytometry. The hearts were stained using haematoxylin and eosin (HE) and Masson's trichome. Myocardial ultrastructure was observed by electron microscopy. Expression of T-bet, GATA-3, IL-4 and IFN-gamma were determined by real-time RT-PCR. The heart was dilated significantly in the C57BL/6 WT + alcohol group and Balb/c + alcohol group compared with the controls. CD4 + CD25 + Foxp3+ Tregs were not statistically different. Masson's trichome staining revealed that fibrosis was more pronounced in the alcohol treated groups than the controls. Fibrosis was more evident in the Balb/c + alcohol group compared to the C57BL/6 WT + alcohol group. Alcohol consumption caused a decrease in the Th1 poalrization and an increase in the Th2 response. Conclusions Chronic alcohol consumption induced a Th2 response within the Th1/Th2 balance. Th2 response is one of the underlying mechanism involved in alcohol-induced cardiac fibrosis.

195. Chronic intake of selective serotonin reuptake inhibitors leads to variations in the expression of 5-HTT, IFN-gamma, and IL-2 genetic message in major depression patients

195. Chronic intake of selective serotonin reuptake inhibitors leads to variations in the expression of 5-HTT, IFN-gamma, and IL-2 genetic message in major depression patients

Influence of allitridin on transcription, expression and function of IL-12 genes in mice infected by murine cytomegalovirus

To investigate whether allitridin could interfere with the effects of murine cytomegalovirus (MCMV) infection on the transcription, expression and function of IL-12 genes in order to further explore the mechanism of allitridin against MCMV. Sixty mice were randomly divided into allitridin treated group, placebo and blank controls. Allitridin was intra-peritoneal injected to mice in treated group once a day with general dosage (25 mg x kg(-1)) at 24 hours after MCMV infection, and the same dosage of physiological saline were given to placebo and blank groups. Four experimental mice were sacrificed at 3, 5, 7, 10, 14 days after treatment (n = 4 per time point), respectively. The expression of IL-12 p70 and IFN-gamma in supernatant of spleen cell cultures were measured by double-antibody sandwich ELISA, and IL-12 p35 and p40 mRNAs in spleen cells were analyzed by RT-PCR. In systemic infection mice, the expression of both IL-12 p70 protein and p35 mRNA significantly increased on day 3 post-infection (pi); then rapidly and markedly decreased on day 5 pi and later. The level of IFN-gamma reached the peak on day 3 pi, then gradually dropped and returned to normal levels during the period of day 10 to 14 pi, and IL-12 p40 mRNA level was persistently and significantly higher after infection. In allitridin treated mice, the levels of IL-12 p70 protein, IL-12 p35 and p40 mRNAs reached the peak on day 3 after treatment (P < 0.05), and then rapidly dropped to the normal levels during the period of 5-14 days. Level of IFN-gamma was also reached the peak on day 3 after treatment; however, it dropped a little on day 5 and then gradually increased and was much higher than those of both placebo and bland controls during the period of day 7 to 14 after treatment (P < 0.01). Allitridin could completely correct the disturbance of expression of IL-12 gene caused by MCMV and persistently promote IFN-gamma expression, which was useful for enhancing the specific cellular immune reactions against CMV and clearance of CMV viruses from host. The result suggests another mechanism of allitridin against CMV.

Modulatory effect of triptolide on differentiation of human Th17 cells

To investigate the effect of triptolide on the differentiation of human Th17 cells. Human peripheral blood mononuclear cells, purified CD4+ T cells and CD4+CD45RA- memory T cells were treated with various concentrations of triptolide in vitro. Cell proliferation was determined by MTT assay. Flow cytometry was used to analyze the intracellular expression of IL-17 and IFN-gamma. Cytokine production of IL-17 and IFN-gamma was measured by ELISA. Cell proliferation, intracellular expression of IL-17 and IL-17 secretion were inhibited by triptolide in a dose-dependent manner. IFN-gamma expression and production were also inhibited by triptolide. Triptolide inhibits the differentiation of human Th17 cell. The observation may indicate at least one of the mechanisms of the immunosuppressive and anti-inflammatory effects of triptolide.

Abstract 5000: Tolerogenic transcription regulator Foxp3 controls the expression of immunosuppressive cytokines in melanoma cells

Abstract Foxp3 is a critical transcription regulator that is found commonly expressed in a subset of immunosuppressive CD4+/CD25+ T cells characterized as regulatory T cells (Tregs) in the immune system. Foxp3 is a member of the forkhead/winged-helix family of transcriptional regulators exhibiting both, activation and repression of transcription. Foxp3 is critically important for the development and function of Tregs and the consequent definition of immune-tolerance. Ectopic Foxp3 expression has been shown to -phenotypically and functionally- convert T cells to Tregs. Recently, we have characterized the expression of Foxp3 in a variety of human and mouse cancerous cells, either in tissue or in cultured cell lines. Although of cardinal importance, the specific role and mechanisms involved in the Foxp3-mediated immunosuppressive activity remains elusive. Thus, we hypothesize that tumor-derived Foxp3 is controlling the transcriptional expression of immunosuppressive cytokines by tumor cells and inducing immunotolerance in the tumor microenvironment. We have tested this hypothesis by interrogating the well-characterized B16-C57BL/6 syngeneic mouse model of melanoma. Foxp3-expressing B16-F0 cells (B16-Foxp3+) where specifically and stably knockdown for Foxp3 expression by lentivirus-mediated shRNA (B16-Foxp3-). In order to determine the specific role and mechanisms of melanoma-derived Foxp3 in controlling the expression of immunosuppressive cytokines generated by melanoma cells, we have evaluated the differential cytokine expression profile between the B16Foxp3+ and the B16-Foxp3- cultured cells using Multiplex Bead Immunoassay, Immunoblotting Assay of Proteins and Semiquantitative RT-PCR. Our results demonstrated that the suppression of expression of Foxp3 in melanoma cells showed a significant downregulation of a selected group of immunosuppressive cytokines, in particular IL-4, IL-6, IL-10, IL-12, TNF-alpha and GM-CSF. In contrast, Foxp3 did not have an appreciable effect on the expression of IL-1, IL-2, IL-5, and IFN-gamma. Altogether, our data suggest the specific role of tumor-derived expression of FOXP3 in the control of the expression of tolerogenic/immunosuppressive cytokines secreted by melanoma cells to the tumor microenvironment. Furthermore, selective suppression of Foxp3 expression and/or activity in cancerous cells may help to develop better and more effective targeted immunotherapy as treatment for malignant melanoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5000. doi:10.1158/1538-7445.AM2011-5000

Abstract 5525: Profiling counter-regulatory and cytotoxic immune pathways with cellular biomarkers in thyroid cancer patients

Abstract Clinical studies suggested an association between thyroid cancer outcome and patient immunologic function. Cytotoxic T-Lymphocyte Antigen 4 (CTLA4) and Programmed Death 1 (PD-1) are strong negative regulators of immune function whose aberrant function has been associated with poor outcomes in patients with solid malignancies. We carried out this study to compare the degree and patterns of expression of CTLA4 and PD-1 in peripheral circulating mononuclear cells in adult patients with thyroid cancer (N=21) compared with adult healthy controls (N=5). We used multiparameter flow cytometry to determine the absolute cell count per ml of blood for the cells of interest. Functional assays of immunologic competence of B-cell, T-cell as well as NK cell subpopulations was assessed by granzyme-B and interferon (IFN) gamma production following PMA/ionomycin stimulation in vitro using standard assay method. Differences in specific cell count between patients and healthy control was assessed by F-test statistics and p-value &amp;lt;0.05 was considered significant. There was no significant difference between cancer patients and healthy controls in the overall numbers of T-cell, B-cell, and NK cell subsets. Similarly, the proportion of CD4, CD8 and NK cell subsets with CTLA4, PD-1, IFN-gamma or Granzyme-B expression with and without PMA/ionomycin stimulation were not significantly different when comparing patients with thyroid cancer to healthy controls. Within the thyroid cancer patient population, however, the distribution of CD4 (+) T-cells, total B-cells, plasma cells, and PD ligand 1-expressing monocytes, macrophages and dendritic cells i.e. CD64 (+) PD-L1 (+) was significantly different between patient with active disease and those in remission. The Granzyme-B (+) CD4 (+) T-cell was significantly lower in patients in remission than in patients with active disease both with and without in vitro activation with PMA/ionomycin (0.08 vs. 0.19 per ml blood × 10⁁6; p=0.03). Intriguingly, functional profiling in a limited number of thyroid cancer patients revealed a markedly impaired ability of CD8 (+) T-cells to produce IFN-gamma and Granzyme B following PMA/ionomycin stimulation. Thus suggesting possible immune exhaustion in this subpopulation of thyroid cancer patients. Overall, our results show a trend toward impaired cytotoxic immune pathway in thyroid cancer patients with active disease burden when compared to patients in remission. A similar trend was observed in comparison to healthy subjects but the small number of control subjects limits statistical significance. Further elucidation of this finding using larger patient sample is clearly warranted and currently ongoing. (Supported by Career Development Award (NCI P50 CA128613) to TK Owonikoko and the Georgia Cancer Coalition Distinguished Scholar Award to DM Shin, SS Ramalingam, FR Khuri, and TK Owonikoko) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5525. doi:10.1158/1538-7445.AM2011-5525

CXCL10 expressing hematopoietic-derived cells are requisite in defense against HSV-1 infection in the nervous system of CXCL10 deficient mice

The chemokine CXCL10 is crucial for the control of viral replication through the regulation of mobilization of antigen-specific T cells to sites of infection. CXCL10 is highly expressed both at sites of inflammation as well as constitutively within lymphoid organs by both bone marrow (BM)-derived and non-BM-derived cells. However, the relative immunologic importance of CXCL10 expressed by these divergent sources relative to HSV-1 infection is unknown. Using mouse chimeras reconstituted with either wild type or CXCL10 deficient mouse BM, we show BM-derived, radiation-sensitive cells from wild type mice were solely responsible for resistance to HSV-1 in the trigeminal ganglia and brain stem. The resistance was not reflected by a deficiency in the recruitment of effector cells to sites of inflammation or expression of chemokines or IFN-gamma and likely results from additional, yet-to-be-determined factors emanating from wild type, BM-derived cells.

Chronic IFN-gamma expression alters the migration pattern of peripheral T cells (160.7)

Abstract We report here on preliminary data from an IFN-gamma(IFN-γ)ARE-deleted Balb/c mouse model developed in our laboratory.This mouse has a 162 nt deletion in the 3’untranslated region of the IFN-γ mRNA to eliminate an AU-rich sequence. The deletion has a positive impact on IFN-γ mRNA accumulation by dramatically increasing its half-life. In the absence of stimulatory factors,these mice express low level of IFN-γ constitutively. IFN-γ ARE-deletion results in a severe phenotype on Balb/c mice. The knockout (KO) and heterozygous (Het) mice are smaller in size than the littermate controls, have a rough coat and a low survival rate after weaning. Additionally, these mice have an enlarged thymus with an increased percentage of mature naïve T cells. The mature thymocytes from Het animals are able to leave the thymus and enter peripheral lymphoid tissues. However, the results from chimera experiments indicate that IFN-γ may impact the thymic environment, thus resulting in abnormal T cell development. In addition, we found the migration pattern of peripheral T cells from Het mice differs from that observed with cells from control animals, further suggesting an abnormality in the Het T cell physiology. Current experiments focus on analyzing changes in the thymic environment and T cell development in newborn mice. In addition, we are analyzing chemokine receptor expression to determine if changes in these receptors may be a mitigating factor in the T cell migration patterns.

1277 OVEREXPRESSION OF THE BIOMARKER MIR-221 REGULATED THE INTERFERON-GAMMA SIGNALLING CASCADE IN PROSTATE CARCINOMA CELLS

You have accessJournal of UrologyProstate Cancer: Basic Research1 Apr 20111277 OVEREXPRESSION OF THE BIOMARKER MIR-221 REGULATED THE INTERFERON-GAMMA SIGNALLING CASCADE IN PROSTATE CARCINOMA CELLS Martin Spahn, Susanne Kneitz, Markus Krebs, Hubertus Riedmiller, and Burkhard Kneitz Martin SpahnMartin Spahn Würzburg, Germany More articles by this author , Susanne KneitzSusanne Kneitz Würzburg, Germany More articles by this author , Markus KrebsMarkus Krebs Würzburg, Germany More articles by this author , Hubertus RiedmillerHubertus Riedmiller Würzburg, Germany More articles by this author , and Burkhard KneitzBurkhard Kneitz Würzburg, Germany More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2011.02.963AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES We previously demonstrated that mir-221 expression is progressively reduced in aggressive prostate cancer and lymph node metastasis and is an independent predictor for cancer specific survival in high-risk prostate cancer. However, up to now it is only purely understood how mir-221 downregulation might be involved in tumor initiation and progression. METHODS We transiently transfected two androgen insensitive PCa cell lines (PC-3 and DU-145) with precursor mir-221 and measured for cell viability, proliferation rates and apoptosis. To analyse the molecular changes in these cells we performed microarray experiments on mRNA isolated from PC-3 cells transfected with precursor mir-221. Upregulated genes involving the IFN-Gamma (IFNg) cascade (e.g. Stat-1 and, IRF 1 and IRF-9) were further analysed by qrt-PCR and western blots. PC3 and Du-145 cells were treated with IFNg to measure cell proliferation, mRNA and protein expression of IFNg cascade members. RESULTS Transfection with pre-mir-221 leads to efficient overexpression of mature mir-221 resulting in biological changes like decreased cell viability and proliferation rates or increased apoptosis. To analyse the molecular changes in these cells we performed microarray experiments on mRNA isolated from PC-3 cells transfected with precursor mir-221. Hierarchical clustering revealed a list of genes of which the expression was elevated. Many of these genes are members of the IFNg cascade (IFNg)(e.g. Stat-1 and IRF1 and IRF-9). Upregulation of genes involved in the IFNg cascade in mir-221 overexpressing cells were confirmed by RT-PCR and Western Blotting. Treatment of PCa cells with IFNg recapitulated some of the aspects of the molecular and phenotypic changes observed in pre-mir-221 transfected PCa cells. However pre-mir-221 transfection did not induce the upregulation of IFNg expression nor could increased IFNg levels be found in conditioned media from treated cells. Moreover we observed a sensitisation of PCa cells for INFg mediated growth inhibition in mir-221 overexpressing cells. CONCLUSIONS From these experiments we concluded that mir-221 overexpression in PCa cells leads to anti-proliferative and pro-apoptotic effects. Moreover we could show that mir-221 overexpression induces the IFNg signalling cascade without treatment with exogenous IFNg or endogenous induced IFNg expression. Our results further demonstrated that mir-221 expression sensitized PCa cells to anti-proliferative effects of IFNg. MiR-221 therefore has potential as a target for new treatment strategies. © 2011 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 185Issue 4SApril 2011Page: e511 Advertisement Copyright & Permissions© 2011 by American Urological Association Education and Research, Inc.MetricsAuthor Information Martin Spahn Würzburg, Germany More articles by this author Susanne Kneitz Würzburg, Germany More articles by this author Markus Krebs Würzburg, Germany More articles by this author Hubertus Riedmiller Würzburg, Germany More articles by this author Burkhard Kneitz Würzburg, Germany More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

S22-02 - The effect of antidepressants on inflammatory markers in animal models of depression

Considering the evidence that pro-inflammatory cytokines play a causal role in depressive illness, the ability of antidepressants to induce anti-inflammatory effects is a subject of considerable interest. In an in vivo context we observe that antidepressants that enhance noradrenaline availability are the most effective anti-inflammatory agents; a fact consistent with the established anti-inflammatory actions of noradrenaline. Specifically, we have observed that noradrenaline reuptake inhibitors (NRIs) inhibit microglial activation and inhibit expression of pro-inflammatory cytokines (IL-1beta and TNF-alpha), iNOS, and inflammatory chemokines (IP-10 and RANTES) in rat brain following a systemic inflammatory challenge. These in vivo anti-inflammatory actions of NRIs are mimicked by in vitro exposure of primary glial cells to noradrenaline, but not by in vitro exposure of glial cells to the drugs themselves. These data suggest that NRIs promote an anti-inflammatory environment in rat brain in vivo by increasing noradrenaline availability at glial cells. We have also observed that even in the absence of any overt inflammation, chronic treatment with the NRI reboxetine promotes an anti-inflammatory phenotype in the CNS characterised by reduced expression of pro-inflammatory cytokine IFN-gamma, and increased expression of the anti-inflammatory cytokine IL-10. Current experiments are focused on the activation of the inflammatory response system in animal models of depression secondary to inflammation. The models are used subsequently to assess the anti-inflammatory effects of antidepressants in vivo.

Histamine regulates relevant murine dendritic cell functions via H4 receptor

Histamine, produced by dendritic cells (DCs) or by other cells of the immune system, may have significant impact on DC activities. We investigated the influence of histamine and histamine H4 receptor (H4R) on some relevant functions of DCs. Histamine significantly decreased the antigen presentation capacity of splenic DCs, and this effect was reversed by a H4R antagonist. Furthermore, enhanced antigen presentation was detected in H4R-/- DCs. Prolonged histamine treatment during DC differentiation stimulated migration, albeit the increase was not significant. H4R-deficient DCs possessed significantly lower migration capacity than their wild-type counterparts. Monitoring in vivo and in vitro DC cytokine production revealed that a H4R agonist in combination with LPS, increased IL-1 beta mRNA expression, and a H4R antagonist reversed this effect. In H4R-deficient mice we detected decreased mRNA expression of some DC-derived cytokines including IFN-gamma and IL-10. Upon CFA stimulation, genotype-dependent differences were found in the expression of IL-6 and IFN-gamma. Our data suggest that H4R plays a crucial role in variety of functions of murine DCs.

The association between HBV genotyping and clinical characteristics and expression of TH1/TH2 cytokines

To explore the association between HBV genotyping and clinical characteristics and expression of TH1/TH2 cytokines. The expression of IL-4 and IFN-gamma was detected with flow cytometry for 102 HBV infections and 48 healthy controls. 50 CHB patients were randomly selected for HBV genotyping with real-time fluorescence PCR assay. Higher expression of IL-4 in peripheral blood was detected in patients with HBV infection than healthy controls (P < 0.001); No significant differences on expression of Th1/Th2 cytokines were observed in CHB patients with different HBV DNA levels or HBeAg status (P > 0.05). There were 34 (68%) patients with genotype B infection and 16 (32%) with genotype C infection. Compared to patients with genotype B infection, the patients with genotype C infection showed higher levels of IL-4 (P = 0.018), and Th1/Th2 ratio decreased,but the difference was not statistically significant (P = 0.2262). The different expression of TH1/TH2 cytokines may elucidate cellular immune response and clinical outcome difference between patients with genotype B infection and genotype C infection.

Bone Marrow Immunological Changes During Treatment with Lenalidomide In Low and Intermediate-1 Risk Myelodysplastic Syndromes with Del(5Q)

AbstractAbstract 2927 Background:Immunological changes have a primary role in the initiation and progression of myelodysplastic syndromes (MDS). Cytokine levels, such as IL-7 and IFN-gamma, are associated with lower-risk disease. Treatment with lenalidomide has proven efficacy in red blood cell (RBC) transfusion-dependent lower-risk MDS patients with del(5q). Lenalidomide exerts anti-angiogenic, anti-proliferative, and pro-erythropoietic effects; in particular, it has been shown that lenalidomide inhibits the proliferation and function of T regulatory cells (Tregs). Finally, MDS patients undergoing lenalidomide treatment experience erythroid responses and suppression of the del(5q) clone. Aims:In a multicenter Italian phase II trial to evaluate safety and efficacy of lenalidomide in primary MDS patients with del(5q) and Low- or Int-1 risk IPSS, we investigated changes in the transcription of cytokines and their receptors during treatment. Methods:The starting dose of lenalidomide was 10 mg p.o once daily on a continuous daily schedule for a maximum of 12 months. Bone marrow (BM) aspirates were obtained on study entry and every 12 weeks. Assays were performed using TaqMan® Low Density Array Fluidic card (TaqMan® Human Array, Applied Biosystems, Foster City, CA, USA) based on Applied Biosystems PRISM® 7900HT comparative ddCT method, according to the manufacturer's instructions. Target gene expression levels were measured in triplicate and normalized against the expression of the 18S housekeeping gene from a BM pool of normal, healthy subjects at all timepoints. Median relative gene expression values in MDS patients were compared to healthy subjects, set as a value of 1. Results:We report data obtained at baseline and after 12 weeks. Informed consent was obtained in all patients. Twenty-seven patients (5 M, 22 F) were evaluated at baseline and after 12 weeks. Mean age was 72 ± 9 years. Mean Hb level was 8.5 ± 0.9 g/dL and 16 patients were RBC transfusion -dependent (requiring at least 4 RBC transfusions in the preceding 2 months). Seven patients had additional cytogenetic abnormalities. Twenty-one patients (80%) experienced erythroid responses by week 12. Significant variations in gene expression of cytokines and receptors were observed during treatment. Genes significantly regulated during lenalidomide treatment (P < 0.05) are shown in the Table. In particular, FAS, IL-7 and FOXP3 gene were generally under-expressed at baseline and significantly increased after 12 weeks. Accordingly, IL7R was over-expressed in all patients at baseline and its expression was significantly reduced during treatment. Furthermore, IFN-gamma expression increased during therapy. Summary:The protein encoded by FAS gene is a member of the TNF-receptor superfamily and its interaction with its ligand leads to apoptosis. Interleukin (IL)-7 is an essential cytokine that promotes the proliferation and survival of B- and T-lymphocyte progenitors. The IL7R gene on chromosome 5 (5p13) codifies for the IL7 receptor, which blocks apoptosis during differentiation and activation of T lymphocytes. It functions, in part, through the induction of the expression of the antiapoptotic protein Bcl-2. The protein encoded by the FOXP3 gene is a member of transcriptional regulators. Defects in this gene are the cause of X-linked autoimmunity-immunodeficiency syndrome. The results of the present study indicate that lenalidomide may act through immunological changes. Further detailed analyses in these patients may provide new insights into the pathogenesis of MDS with del(5q) and the long-term effects of lenalidomide treatment on immunological changes in BM cells.Table:Changes in gene expression of bone marrow cytokines during lenalidomide treatment in MDS del5q patients.GenesBaseline gene expression Median (IQ range)Gene expression after 12 weeks Median (IQ range)p-valueFAS0.04ü(0.02ü–ü0.60)23.68ü(0.09ü–ü586.51)<0.0001Gamma-IFN0.69ü(0.23ü–ü7.86)45.23ü(9.87ü–ü1138.67)0.001IL-12A0.35ü(0.15ü–ü7.35)3.84ü(0.27ü–ü202.29)0.030IL-12B0.31ü(0.08ü–ü6.64)22.75ü(0.37ü–ü2.70)0.002IL-70.29ü(0.06ü–ü0.86)4.94ü(0.19ü–ü25.07)0.012Il-7R2305.10ü(1081.50ü–ü6261–73)0.12ü(0.00ü–ü2.80)<0.0001IRF70.12ü(0.04ü–ü0.97)7.98ü(0.25ü–ü1.42)0.005FOXP30.83ü(0.35ü–ü2.02)7.22ü(3.71ü–ü13.20)<0.0001 Disclosures:Oliva:Celgene: Consultancy.