Robert L. Elliott

Differential Gene Expression Analysis Reveals Generation of an Autocrine Loop by a Mutant Epidermal Growth Factor Receptor in Glioma Cells

The epidermal growth factor receptor (EGFR) gene is commonly amplified and rearranged in glioblastoma multiforme leading to overexpression of wild-type and mutant EGFRs. Expression of wild-type EGFR ligands, such as transforming growth factor-alpha (TGF-alpha) or heparin-binding EGF (HB-EGF), is also often increased in gliomas resulting in an autocrine loop that contributes to the growth autonomy of glioma cells. Glioblastoma multiformes express a characteristic EGFR mutant (EGFRvIII, de 2-7) that does not bind ligand, signals constitutively, and is more tumorigenic than the wild-type receptor. However, the downstream signals that mediate this increased tumorigenicity are not well understood. We hypothesized that signals induced specifically by EGFRvIII and not the wild-type receptor are more likely to mediate its increased tumorigenic activity and examined the gene expression profiles resulting from inducible expression of comparable levels of either wild-type EGFR or EGFRvIII in a U251-MG glioma cell line. Expression of EGFRvIII resulted in specific up-regulation of a small group of genes. Remarkably, all these genes, which include TGFA, HB-EGF, EPHA2, IL8, MAP4K4, FOSL1, EMP1, and DUSP6, influence signaling pathways known to play a key role in oncogenesis and function in interconnected networks. Increased expression of EGFRvIII-induced genes was validated by real-time PCR. The mutant receptor does not bind ligand, and EGFRvIII-induced expression of TGF-alpha and HB-EGF suggests that EGFRvIII plays a role in generating an autocrine loop using the wild-type EGFR in glioma. It also raises the possibility that EGFRvIII may signal, at least in part, through the wild-type receptor. Indeed, we show that inhibiting the activity of HB-EGF, a potent mitogen, with neutralizing antibodies reduces cell proliferation induced by expression of EGFRvIII. This suggests that the EGFRvIII-HB-EGF-wild-type EGFR autocrine loop plays an important role in signal transduction by EGFRvIII in glioma cells. We also show by immunohistochemistry that HB-EGF expression correlates with the presence of EGFRvIII in glioblastoma multiforme. Thus, our study provides a new insight into oncogenic signaling by EGFRvIII and improves our understanding of how autocrine loops are generated in glioma.

Human Leukocyte Antigen G Expression in Breast Cancer: Role in Immunosuppression

Human leukocyte antigen G (HLA-G) is an immunotolerant nonclassical major histocompatibility complex Class Ib molecule. It is expressed by trophoblastic placental cells during pregnancy to protect the fetus from maternal alloreactivity. HLA-G is overexpressed in tumors and involved in cancer immune evasion. Reverse transcription-polymerase chain reaction and immunohistochemistry (IHC) were used to examine HLA-G expression in normal mammary and breast cancer cell lines and normal and human breast cancer tissues. Reverse transcription-polymerase chain reaction confirmed that normal epithelial MCF-12A cells had no HLA-G mRNA expression, whereas cancer cell lines MCF-7, T47D, and MDA-MB-231 and NCI/Adr-Res had various levels of HLA-G mRNA expression. Twelve (12) normal and 38 breast cancer tissues were examined by IHC. Fifty-eight (58) percent (22/38) of cancers had medium to strong staining to HLA-G, whereas only 8% (1/12) of normal breast tissues had medium to strong staining, and the difference was significant (p < 0.05). HLA-G staining was found in the membranes and cytoplasm of cancer cells. In conclusion, breast cancer cells overexpress HLA-G mRNA and protein, and this probably contributes to immune evasion.

Cancer risk assessment with a second-generation infrared imaging system

Infrared imaging of the breasts for breast cancer risk assessment with a second generation amber indium antimonide focal plane staring array system was found to produce images superior to a first generation Inframetrics scanning mercury cadmium telluride system. The second generation system had greater thermal sensitivity, more elements in the image and greater dynamic range, which resulted in a greater ability to demonstrate asymmetric heat patterns in the breasts of women being screened for breast cancer. Chi-square analysis for independence of the results from 220 patients with both the scanning and focal plane infrared imaging systems demonstrated that the results from the two systems were strongly associated with each other (p equals .0001). However, the improved image from the second generation focal plane infrared imaging system allowed more objective and quantitative visual analysis, compared to the very subjective qualitative results from the first generation infrared imaging system. The improved image also resulted in an increase in the sensitivity for asymmetric heat patterns with the second generation focal plane system and yielded an increase in the percentage of patients with an abnormal asymmetric infrared image of the breasts from 32.7% with the scanning system to 50.5% with the focal plane system. The greater sensitivity and resolution of the digitized images from the second generation infrared imaging system has also allowed computer assisted image analysis of both breasts, breast quadrants and hot spots to produce quantitative measurements (mean, standard deviation, median, minimum and maximum temperatures) of asymmetric infrared abnormalities.

Abstract 2892: Vaccination with a therapeutic cancer vaccine containing prostate specific antigen and the biological adjuvants IL-2 and GM-CSF results in reduced serum PSA in prostate cancer patients

In 1997 we initiated a phase I/II clinical trial of a therapeutic prostate cancer vaccine. We have enrolled 12 biopsy confirmed prostate cancer patients in this study and 11 finished the initial course of 6 intradermal vaccinations containing prostate specific antigen (50 μg) and biological adjuvants (IL-2, 20,000 units, and GM-CSF, 16.7 μg) at 0, 1, 2, 6, 10 and 14 weeks. During this study the prostate cancer patients received no other concurrent therapy (surgery, hormone, radiation, radioactive seeds, chemotherapy). One of the 12 patient9s PSA rose from 7.6 to 13.7 after the fourth vaccination and he (patient #8) withdrew from the study to seek other therapy. In the remaining 11 prostate cancer patients serum PSA concentrations were determined before initiating vaccination and 3-4 weeks after the 6th vaccination. There was a decrease in the PSA levels in 8 of 12 of the prostate cancer patients after 6 vaccinations. One of the patients (patient #3), whose PSA had dropped from 6.8 to 6.4 and had previously received radiotherapy (the only patient previously treated), elected to withdraw from the study and underwent radical prostatectomy. Nine of the original 12 patients have received monthly 3 intradermal IL-2 injections (11 million units) alternated with 3 further vaccinations for the 6 months following the initial vaccinations, and 8 patients have been followed from 18 to 92 months. The mean PSA values for the 8 patients still being followed without additional therapy were 5.8 initially, decreasing to 4.1 after 6 vaccines, 3.7 after 12 vaccines and is 4.7 after a mean follow-up of 49 months (median follow-up of 45.5 months). These promising results have led to the creation of the U.S. Navy Cancer Vaccine Program with a Phase I clinical trial at the Veterans Administration Medical Center San Diego/UCSD Medical School. Citation Format: Jonathan F. Head, Robert L. Elliott. Vaccination with a therapeutic cancer vaccine containing prostate specific antigen and the biological adjuvants IL-2 and GM-CSF results in reduced serum PSA in prostate cancer patients. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2892. doi:10.1158/1538-7445.AM2014-2892

Whole cell preparations of MCF-7 breast carcinoma cells are more active than lysed cell preparations in lymphocyte blastogenesis assays (LBAs) of breast cancer patients

2596 Background: We have previously reported that whole cell preparations of breast carcinoma cells from either an autologous or allogeneic source (MCF-7 cells) could produce immune responses, increased lymphocyte proliferation in a LBA, in breast cancer patients vaccinated with tumor cells, tumor antigens and two biological adjuvants (IL-2 and GM-CSF). We also showed that after vaccination with the tumor antigens (CEA, CA 15-3 and CA125), there were significant increases in the proliferation of the breast cancer patients lymphocytes in response to the antigens in the LBA. The present study was undertaken to determine if tumor cell lysates would be more antigenic than the whole cell preparations.nnnMETHODSnMCF-7 cells were lysed by two cycles of freeze thawing and a sample was tested in cell culture for viability. The lysate was then tested in LBAs and the results compared to those from whole cell preparations of MCF-7 cells (detached from cell culture plate, allowed to regenerate surface antigens while being agitated, and treated with mitomycin C to prevent further proliferation).nnnRESULTSnWe found that there was significantly less activity in the lysate (38% to 81% less) than the whole cell preparation in 6 of the LBAs from 9 breast cancer patients. However, the lysate was much more active in 2 of the patients LBAs (280% and 350% increases). One patient showed no difference between the two preparations.nnnCONCLUSIONSnWhole cell preparations of allogeneic MCF-7 cells have immunologic activity in more breast cancer patients than cell lysates, as determined by LBAs. However, a small but significant proportion (20 to 25%) of patients have a significantly greater immunologic response to the cell lysate. Further investigation of the pre-vaccine LBA activity of the whole cell and cell lysate as they relate to immunologic response to our breast cancer vaccine is warranted to determine if the cell lysate should be added to the vaccine. No significant financial relationships to disclose.

Abstract 1664: Inhibition of 4T1 mammary tumor growth in BALB/c mice by subcutaneous and intraperitoneal injection of a 4T1 whole cell vaccine containing IL-2 and GM-CSF as adjuvants

Developing animal tumor models for human cancer vaccines creates a tool to investigate the mechanism of action, variations in formulation, dosing schedules, and combinations with other forms of cancer therapy. In this study we developed a mouse whole cell mammary cancer vaccine model with both subcutaneous and intraperitoneal injection of the vaccine. The vaccine, containing 4T1 mouse mammary cancer cells (1,000,000 cells), IL-2 (0.2 µg) and GM-CSF (0.1 µg) in a total volume of 150 µl, was injected either subcutaneously into the backs of BALB/c mice or intraperitoneally. There were six injections of the vaccine (weeks 1, 2, 3, 7, 11, 15) and one week after the last injection 100,000 4T1 cells from cell culture in a volume of 100 µl were injected into the scapular region of the mice. At 22 days post 4T1 tumor transplantation there was a 37% inhibition of the growth of the 4T1 tumor in the mice receiving the subcutaneous injection of the vaccine and a 42% inhibition of the growth of the 4T1 tumor in the mice receiving the intraperitoneal injection of the vaccine. The inhibition of 4T1 mammary tumor growth in this mouse model with subcutaneous injection of a whole cell vaccine gives further support for the previously reported efficacy in a Phase 1/2 clinical trial of our therapeutic breast cancer vaccine containing autologous and allogeneic breast cancer cells in the adjuvant setting. The inhibition of 4T1 tumor growth with intraperitoneal injection suggests a delivery method that may be applicable to ovarian cancer and cancer patients with malignant ascites. Citation Format: Jonathan F. Head, Jeffrey T. Phillips, Xianpeng Jiang, Robert L. Elliott. Inhibition of 4T1 mammary tumor growth in BALB/c mice by subcutaneous and intraperitoneal injection of a 4T1 whole cell vaccine containing IL-2 and GM-CSF as adjuvants [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1664. doi:10.1158/1538-7445.AM2017-1664

Abstract A048: Phase 1 clinical trial of a therapeutic prostate cancer vaccine containing PSA/IL-2/GM-CSF in PSA defined biochemical recurrent prostate cancer patients

Immunotherapy for cancer has had two main approaches that have lead to clinical applications. The first is stimulating immune responses to tumor cells with cytokines or cellular immunotherapy and the second is blocking tumor immune evasion and the associated inhibition of T-cell activation with antibodies to the CTLA-4 receptor, PD-1 receptor or PD-L1. We have taken a different approach and have developed therapeutic cancer vaccines that are a combination of tumor antigens (whole cells or proteins) with biological adjuvants (the cytokines IL-2 and GM-CSF). This study is a Phase 1a/1b clinical trial of a PSA/IL-2/GM-CSF vaccine in recurrent prostate cancer in hormone-naive and hormone-independent patients. Major inclusion criteria include adenocarcinoma of the prostate, rising serum PSA and no measurable disease. Phase 1a examines the rate of dose limiting adverse events (DLAEs) in an initial course of 6 vaccinations (“induction vaccination”). The Phase 1b examines the rate of DLAEs with a continued course of an additional 6 vaccinations (“maintenance vaccine”). All patients will receive intradermal injections of the PSA/IL-2/GM-CSF vaccine at weeks 1, 2, 3, 7, 11, and 15. In an additional 28 patients the six maintenance vaccines will alternate IL-2 and the complete vaccine (PSA/IL-2/GM-CSF) at weeks 23, 27, 31, 35, 39 and 43. To date, twelve of twenty patients in the Phase 1a portion of the trial have received at least one vaccine injection and ten patients have received all 6 vaccines. Seven of the ten patients that have received 3 vaccines had increased responses to PSA in a lymphocyte blastogenesis assay and five of the nine patients had an increase in their response after 6 vaccines. None of the patients vaccinated in the Phase 1a portion have had a DLAE and enrollment continues in the Phase 1a. Citation Format: Jonathan F. Head, Gregory A. Daniels, Michelle McKinney, Weg Ongkeko, Jessica Wang-Rodriguez, Kyoko Sakamoto, Robert L. Elliott. Phase 1 clinical trial of a therapeutic prostate cancer vaccine containing PSA/IL-2/GM-CSF in PSA defined biochemical recurrent prostate cancer patients. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A048.

Vaccination of prostate cancer patients with a therapeutic vaccine containing prostate specific antigen (PSA) and the biological adjuvants IL-2 and GM-CSF results in reduced serum PSA

Autoimmune diseases such as systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis and others affect millions of americans. The polymorphism rs2004640, found in the Interferon regulatory factor 5 (IRF5) promoter has been found to be a risk factor for several autoimmune diseases. We sought to define functional molecular effects due to this risk factor in cells from healthy individuals with the risk factor compared to those without. In cells with this polymorphism, we found >2-fold higher levels of IRF5 mRNA and protein (p<0.05). IRF5 is proapoptotic, so we expected higher levels of IRF5 would lead to increased apoptosis. Apoptosis levels were found to be 2.1 fold higher in risk cells (p=0.012). Since the polymorphism allows use of the IRF5 alternate first exon 1B, we analyzed four potential IRF5 promoters using ChIP-Seq data (ENCODE database) and the FactorBook database to define transcription factor binding sites. From this analysis, a p53 binding site was found only on the promoter for exon 1B. Further analysis measured the proportion of usage of each exon using qPCR. Usage of exon 1C was 2.3-fold lower (p=0.013) and 1D was 3.6-fold lower (p=0.017) in risk cells. We discovered 5 new splice variants of IRF5 mRNA, three of which used the exon 1B promoter. Each new variant was missing part of an instability domain, which may affect protein stability. These changes in expression, apoptosis and splicing help to explain how this polymorphism affects cellular and molecular processes and contributes to the development of autoimmune disease.T cells play a critical role in the pathogenesis of autoimmune diseases. Differentiation of Th17 cells requires the nuclear receptor RORγt, and inhibition of RORγt by small molecule inhibitors prevents Th17 cell differentiation in vitro. RORγt inhibitors also modulate RORγt activity in vivo as demonstrated by a significant reduction in the severity of experimental autoimmune encephalomyelitis. However, the effect of RORγt small molecule inhibitors on the in vivo generation of Th17 cells has not yet been clearly demonstrated. Here we report a novel, potent, and selective RORγt inverse agonist TMP778 and demonstrate its role in the in vivo differentiation of Th17 cells. TMP778 blocks both human and mouse Th17 cell differentiation in vitro. In vivo, administration of TMP778 to C57BL/6 mice immunized with myelin oligodendrocyte peptide 35-55 (MOG3555) blocked MOG-specific IL-17A expression. In addition, expression of Th17 signature genes (Il17a, Il17f, Il22, and CCL20) as well as Ifnγ was inhibited. Further, IL-17-producing CD4+ T cells were significantly reduced in IL-17-IRES-GFP transgenic mice administrated TMP778 after immunization with MOG35-55. Thus, our studies demonstrate that RORγt inverse agonist TMP778 suppress Th17 differentiation in vivo. Jianfei Yang, J Clin Cell Immunol 2013, 4:5 http://dx.doi.org/10.4172/2155-9899.S1.013A is a term used for a complex group of neurodevelopmental disorders characterized by deficits in communication, social skills and the presences of repetitive stereotyped behaviors. Although there has been extensive research into the etiology of autism, little is known. Genome wide association studies have only identified about 5-10% of the genetic risk after studying thousands of autism subjects. Natural killer-cells (NK-cells) are a subset of lymphocytes with the inborn ability to produce cytokines and kill target cells without prior sensitization. They recognize the lack of HLA proteins on the surface of virally infected and transformed cells (missing self ). Among the surface receptors on NK-cells are killer-cell immunoglobulinlike inhibitory or activating proteinreceptors (KIR) that are encoded by genes in the leukocyte receptor complex (Chromosome 19q13/4). The ligands that bind to these receptors are cell-surface HLA class I proteins encoded by genes in the class I region (Chromosome 6). KIR genes and their cognate HLA ligands have been associated with several autoimmune diseases and there is evidence that NK-cells activity is associated with autism. In this presentation, I will present data that suggests a strong association with certain activating KIR genes and their cognate HLA genes in the Caucasian autism population. Anthony R. Torres, J Clin Cell Immunol 2013, 4:5 http://dx.doi.org/10.4172/2155-9899.S1.013H contacts (HHCs) of patients with tuberculosis (TB) are at higher risk of infection as well as the development of active disease. Longitudinal tracking of antigen-specific cytokines with the exposure may significantly help in understanding the dynamic changes in cytokine patterns associated with disease establishment. The aim of the present study is to investigate the role of candidate cytokines stimulated with r32-kDa antigen of M.bovis BCG (Ag85A-BCG) in Active Pulmonary Tuberculosis (APTB) patients, their HHC and Healthy Controls (HC) were assessed at 0, 4, 6, and 12 months after exposure. The in vitro T cell assays, ELISA of (Interferon gamma -IFN-γ), (Tumour Necrosis factor alpha-TNF-α) and (InterleukinIL-4) cytokines of culture supernatants were studied in APTB (n=50), HHC who were tuberculin-Purified Protein Derivative (PPD) skin test positive (n=50) and Healthy Controls (HC) n=50, were studied. The mean proliferative responses of stimulation cells was found to be significant between APTB patients at 4M, 6M, HHC at 4M compared to HC (2±0.55,1.061±0.508,0.99±0.3381.518±0.909 at p<0.02,0.01&0.03) respectively. The mean IFN-γ levels were significantly low in Pts/HHC compared to HC (24.5±19.8/33.55±25.44&107.5±59.0)at p<0.0001. APTB Pts at 0M compared to 4M &6M (24.5±19.8,64.13±43.88&81.97±56.22) at p<0.013&0.0008 and HHC at 0M compared to 4M (33.55±25.44&71.6±56.46) at p<0.031.TNF-α levels were significantly low in Pts at 0M compared to 12M(22.82±20.71&40.83±19.09) at p<0.02. IL-4 levels were significantly high at 0M in Pts and HHC compared to HC (7.46±5.826, 7.495±5.568&5.405±4.639) at p<0.01, 0.007. The ratios for IFN-γ/IL-4 and TNFα/IL-4 were significantly increasing from 0-6 months. Five APTB pts were turned into relapse during the follow up, 2 at 4th month and 3 at 6th month Whereas in HHC IFN-γ & IL-4 levels were decreasing during the follow up from 0-6 months. One of HHC developed the disease at 4th month of follow up. r32Kda M.bovis BCG antigen seems to be immunogenic, stimulating protective immune responses which may help us to assess the treatment outcome. In conclusion, it is important that there is a need to elucidate the mechanism of Th1 and Th2 responses in understanding recurrent TB or susceptibility to disease and also help in identifying the HHC by monitoring their immunological status who are at high risk. Suman Latha G et al., J Clin Cell Immunol 2013, 4:5 http://dx.doi.org/10.4172/2155-9899.S1.013I immune system acts as the first-line of host defense against infection by microbes to activate anti-microbial responses for elimination of invading pathogens. Recent rapid progress in studies on innate immunity has facilitated the identification of various pattern recognition receptors (PRRs), which sense pathogen-derived molecular patterns (PAMPs) and evoke robust innate immune responses through the gene induction of proinflammatory cytokines and type I interferons (IFNs). Particularly, during viral infection, virus-derived nucleic acids are mainly recognized as viral PAMPs by nucleic acid sensors. Among these, a DExD/H-box RNA helicase RIG-I, is known to be a key cytosolic RNA sensor that has an important role in triggering responses to many viruses, such as influenza A virus, measles virus, hepatitis C virus, most of which are causative agents for infectious diseases in human. Recently, our group has identified the poly(ADP-ribose) polymerase-13 (PARP-13) shorter isoform “ZAPS” as a potent stimulator of the RIG-I-mediated signaling. ZAPS interacts with RIG-I to promote its activity, leading to robust activation of IRF-3 and NF-kappaB transcription factors. We also found that ZAPS contributes to RIG-I-mediated antiviral activity during influenza virus infection. On the other hand, such innate immune signals are often targeted by viral proteins to evade host immune system. In our current study, we demonstrate that ZAPS is involved in innate immune evasion of influenza virus. Our data indicate that influenza virus expresses a viral protein to competitively inhibit the interaction of ZAPS with RIG-I. In addition, a study with ZAPS transgenic mice also revealed an important role of ZAPS in antiviral defense against influenza virus in vivo. These results may provide a therapeutic insight for the control of viral infection. Akinori Takaoka, J Clin Cell Immunol 2013, 4:5 http://dx.doi.org/10.4172/2155-9899.S1.013T forkhead box P3 (FOX P3) is a major transcriptional regulatory factor required for the development and functioning of T-regulatory cells (CD4+, CD25+, T-cells). Through interaction with the nuclear factor of the activated T-cells (NFAT), the FOX P3 molecule controls the functioning of the regulatory T-cells. In this presentation we analyze the association of two Single Nucleotide Polymorphisms (SNPs) in the promoter region of the FOX P3 gene -3279C/A and -2383C/T with Graves disease and Hashimoto’s thyroiditis respectively. An important feature of these two autoimmune diseases is that a predominant Th2 type of immune response to thyroid antigens (eg. Anti-TSHR antibodies) is reported in Graves disease contrary to a predominant Th1 response in Hashimoto’s thyroiditis. This indicates a profound influence of the genotypes of -3279C/A and -2383C/T SNPs of FOX P3 gene in determining the pattern of immune response (Th1 / Th2). The role of the above mentioned polymorphisms in affecting clonal expansion/functional efficiency of T regulatory cells and thus resulting in diverse patterns of immune response in autoimmune thyroid disorders will be discussed in combination with genotypes of certain proand anti-inflammatory cytokines. Ethnic differences in the association of genotypes of these SNPs and variations in the frequencies of their genotypes / alleles will also be discussed. Mohammed Ishaq, J Clin Cell Immunol 2013, 4:5 http://dx.doi.org/10.4172/2155-9899.S1.013A of sunshine a day keeps gestational diabetes away? It is established that vitamin D the sunshine vitamin, specifically the active metabolite 1,25(oH)(2)D is involved in homeostasis of bone structure-its mineralization and formation and also the metabolism of calcium and phosphorus. However it is emerging that this metabolite also has effect on other important physiological processes in the body at a cellular level including autoimmune, and also, appear to play a role on the sensitivity of insulin. This report will attempt to evaluate this assumption as well as the notion or possibility of incorporating as part of routine antenatal care in pregnant women in particular those at risk of gestational diabetes. The report will discuss current practices research not only discuss current practices , relevant research as well as metabolic, physiological, biochemical basis Susan Okafor, J Diabetes Metab 2013, 4:6 http://dx.doi.org/10.4172/2155-6156.S1.022Purpose: The nature of heightened endotoxin sensitivity state observed in Familial Mediterranean fever (FMF) at present remains unknown. To assess the possibility that IL-10 plays a role in setting inflammatory threshold we studied the IL-10 production by monocytes and dendritic cells and endotoxin tolerance induction in FMF patients. Methods: 46 attack free FMF patients included in this study. The production of IL-10 by NLRor TLR-agonists stimulated monocytes and dendritic cells assayed either by conventional ELISA and flow cytometry. Versatility of monocytes studied by measuring the production of IL-10 and IL-1β after stimulation by proand anti-inflammatory agents, and after stimulation arrest or a further counter stimulation. Monocyte endotoxin tolerance and cross-tolerance induction assayed by measuring the production of IL-1β, IL-10, TNF-α and IFN-γ after pre-stimulation by NLRor TLR-ligands and after re-stimulation with LPS. Results: In FMF patients we observed down-regulation of circulating CD36+ peripheral blood lymphoid cells but not monocytes, constitutively producing IL-10. The production of IL-10 by TLRand NLR-agonists stimulated monocytes and dendritic cells is declined in FMF patients. Monocytes isolated from FMF patients failed to switch from a pro-inflammatory activated state to antiinflammatory phenotype and still produce IL-1β but not IL-10, which cause impaired endotoxin tolerance and cross-tolerance induction. The IL-10 production and endotoxin tolerance induction by monocytes and dendritic cells restored by NOD2ligand MDP and colchicine treatment. Conclusion: Reduced IL-10 production associated with impaired setting of feedback inhibition of inflammatory response and caused impaired resolution of inflammation and endotoxin tolerance induction. Tigran K. Davtyan, J Clin Cell Immunol 2013, 4:5 http://dx.doi.org/10.4172/2155-9899.S1.013I has been long known that cytokines play important role in cancer initiation, development and progression. Until recently, the lack of suitable high-content and high-throughput approaches has hindered progress in understanding the role of complex cytokine networks in cancer. Since their introduction in 2001, cytokine antibody arrays, which have the ability to detect hundreds of cytokines simultaneously in highly sensitive, high-throughput format, significant advances have been made in our understanding of the role of tumor-stromal interactions and chronic inflammation in tumorigenesis, angiogenesis and metastasis, as well as the elucidation of several cytokine-driven mechanisms of cancer drug resistance in tumor cells. This presentation will discuss cytokine antibody array technologies and their recent contributions toward a better understanding of the mechanisms of cancer hallmarks. Ruo-Pan Huang, J Clin Cell Immunol 2013, 4:5 http://dx.doi.org/10.4172/2155-9899.S1.013T rodent T-cell-dependent antibody response (TDAR) assay is used to assess the effect of candidate therapeutic agents on the immune system by measuring primary and secondary IgM and IgG antibody responses to exogenous antigen challenge. TDAR responses require intact function of multiple immune cells including antigen presenting cells and T and B lymphocytes, as well as a cytokine-dependent isotype class switch from IgM to IgG, resulting in production of an antigen-specific antibody response. Alterations in the amount of antibody produced therefore can reflect effects on any or all of the cell populations involved in TDAR. TDAR is commonly used in preclinical drug development especially where increased cause for concern exists (ICH guideline S8). Development of combination therapy that engages multiple targets impacting the immune system poses unique opportunity for increased efficacy but also unique risk for increased immunotoxicity. For this work, a mouse TDAR evaluating primary and secondary KLH antibody responses in a KLH-Specific IgM and IgG sandwich enzyme-linked immunosorbent assay (ELISA) was used to assess immunotoxicologic potential of multiple single and combination therapies. Combination therapy did not result in enhanced TDAR immunotoxicity compared to single therapy alone for the molecules evaluated. Tier 1 immunotoxicology assessments similar to those in standard toxicity studies were added to the TDAR assessment. Together, our data support the use of the TDAR assay for early safety assessment of potential combination therapies. Amy L. Volk, J Clin Cell Immunol 2013, 4:5 http://dx.doi.org/10.4172/2155-9899.S1.013P is a common mechanism by which the mediators of innate immunity can fight off microbial infections. Our ongoing research demonstrates that human herpesviruses may have evolved a common mechanism to avoid phagocytic degradation by exploiting a phagocytosislike entrapment to gain entry into the cells of the eye. The three representative herpesviruses included in our study are herpes simplex virus-1 (HSV-1) that causes corneal keratitis, cytomegalovirus (CMV), which is associated with retinitis in immunocompromised individuals and the third herpesvirus, human herpesvirus-8 (HHV-8), is related to the pathogenesis of Kaposis sarcoma, a common AIDS-related tumor of eyelid and conjunctiva. Using laser scanning confocal microscopy imaging, we have found that successful infection of ocular cell types by all the three viruses, belonging to three divergent subfamilies of herpesviruses, is facilitated by induction of F-actin rich membrane pseudopods. Inhibitors of F-actin polymerization and pseudopod formation, cytochalasin D and latrunculin B, show strong efficacy in stopping the infection by all three viruses. We also found that an identical inhibition was seen by preventing phosphoinositide 3 kinase signaling, which is required for the phagocytosis of microbes. Transmission electron microscopy data using human corneal fibroblasts for HSV-1, human retinal pigment epithelial cells for CMV, and human conjunctival epithelial cells for HHV-8 provide further support to our argument that pseudopod-like membrane protrusions facilitate virus uptake by the ocular cells. Our findings suggest a novel mechanism by which the nonprofessional mediators of phagocytosis can be infected by human herpesviruses. Deepak Shukla, J Clin Cell Immunol 2013, 4:5 http://dx.doi.org/10.4172/2155-9899.S1.013I regulatory factor 5 (IRF5)is proapoptotic and has polymorphisms that confer risk for autoimmune disease. IRF5 has four first exon choices: 1D, 1A, 1B, and 1C respectively and the promoter region of exon 1A contains a CGGGG indel which is a risk factor for: Systemic lupus erythematosus, Sjogrens disease, Multiple Sclerosis, Crohns disease, and ulcerative colitis as determined by genome wide association studies (GWAS). The presence of this indel changes transcription factor binding in the promoter and correlates with higher levels of IRF5 as transcription levels are dependent on a gene’s first exon. Additionally, based on mRNA testing the 1A exon is also translated most efficiently. Thus the 1A risk indel may contribute to an increase in apoptosis through upregulation of IRF5 by increased transcription and efficient translation. To determine which transcription factors would bind to IRF5’s promoter, we used ChIP-sequencing data (ENCODE database).This data was used in conjunction with the FactorBook database to define where conserved genomic sequences where transcription factors are likely to bind in the promoter regions of IRF5. Exons 1A and 1D were found to contain putative PU.1 and NFkB binding sites. IRF5’s four promoters were cloned into luciferase plasmids to determine promoter activity for each exon using immune and epithelial cells. Imiquimod, a Toll-like receptor 7 ligand, and etoposide were used to activate the promoters. As a result of imiquimod treatment IRF5 levels were doubled (p<0.001), due to increased expression of exon 1A (2.2 fold, p=0.03) and 1D (2.8 fold, p=0.03), although when untreated there is less exon 1D usage in cells with the risk indel. IRF5 itself is a transcription factor for CCR7, one of its downstream targets, yet the risk indel is more likely to turn down CCR7 which suggests that it may be acting as a repressor. Understanding polymorphisms in the promoter regions of IRF5 and the effect that transcription factors play in the activity of these promoters is critical to determining how IRF5 is regulated in those with risk factors for autoimmune disease. Rodney Till, J Clin Cell Immunol 2013, 4:5 http://dx.doi.org/10.4172/2155-9899.S1.013A of innate immune components such as Toll-like receptor (TLR)s during diseases such as infectious diseases, cancer, metabolic diseases and liver disorders are associated with disruptions in metabolism and clearance of drugs. Impairment of drug metabolism/clearance can cause adverse effects of drugs. Thus, understanding the mechanism by which TLRs disrupt drug metabolism is important to develop effective strategies to prevent undesirable effects of drugs in patients with activated immune responses. Drug disposition is regulated by drug metabolizing enzymes (DMEs) and transporters primarily present in the liver. We have previously shown that activation of TLRs reduce the expression of DME/transporter genes. Expression of these genes is regulated by basal transcription factors as well as by nuclear receptors which bind to the promoters of these genes. We hypothesize that activation of TLRs recruit downstream adaptor proteins, leading to induction of cell-signaling components (c-Jun N-terminal kinase (JNK) and NF-κΒ) to reduce the expression of nuclear receptors leading to reduced expression of DMEs/transporter genes. We determined the role of the bacterial receptors, TLR2 (for gram-positive bacteria) and TLR4 (for gram-negative bacteria), their downstream adaptor proteins (TIRAP and TRIF), cell-signaling components and nuclear receptors in the regulation of DMEs/transporters. Pharmacokinetc and pharmacodynamic studies with clinically-relevant medications were also performed. These studies establish the role of innate immune components as novel regulators of drug metabolism/ clearance. These regulators can be targeted to prevent or reduce the undesirable effects of drugs in patients. Romi Ghose, J Clin Cell Immunol 2013, 4:5 http://dx.doi.org/10.4172/2155-9899.S1.013Identifying the immune correlates of protection is a key aspect of developing vaccines against infectious pathogens. We designed a universally applicable strategy to profile the antibody (Ab) repertoire of protected vaccine recipients, using recombinant phages encoding random peptide libraries. The novel approach, termed “protection-linked (PL) biopanning,” uses subtractive biopanning and probes Ab paratopes of protected vaccinees (“winners”) versus those with vaccine failure (“losers”). As proof-of-concept, we screened plasma samples from vaccinated macaques that had completely resisted multiple mucosal challenges with CCR5-tropic simian-human immunodeficiency viruses (SHIVs). The animals had been immunized with multimeric HIV-1 gp160 (Env), HIV-1 Tat, and SIV Gag-Pol particles. PL biopanning yielded a panel of approximately 100 recombinant phages that specifically reacted with Abs from “winners” and partially protected macaques but not Abs from “losers” or non-vaccinated, virus-challenged controls. Only about half of the phagotopes selected revealed amino acid homologies with HIV-1 Env (Env mimotopes). Expanding the sequence analysis to include non-Env immunogens revealed an unexpected additional humoral correlate of protection: the neutralizing epitope of HIV-1 Tat. Functional assays confirmed the induction of neutralizing anti-Tat Abs in macaques with full or partial protection but not in those with vaccine failure. Our data suggest that Tat should be included in multicomponent HIV-1 vaccines – and highlight the power of the new PL-biopanning strategy to identify Ab responses with significant association to vaccine protection, regardless of the mechanism(s) or targets of the protective Abs. PL biopanning is unbiased with regard to pathogens or disease model, making it a universal tool. Biography: Ruprecht obtained her Ph.D. in Human Genetics from Columbia University, NY, and her M.D. from the University of Miami, FL. After training in internal medicine at UCLA and hematology-oncology at the Memorial Sloan-Kettering Cancer Center, NY, she joined the Dana-Farber Cancer Institute and Harvard Medical School, where she became a tenured professor of medicine. She recently joined the Texas Biomedical Research Institute/Southwest National Primate Research Center, where she plans to expand the AIDS Research Program. She is internationally known for her studies on AIDS vaccine development and lentiviral pathogenesis in primate models, especially mucosal virus transmission and its prevention.C of ligand-engaged cytotoxic T lymphocyte antigen-4 (CTLA-4) to the T cell receptor (TCR) during the early phase of T cell activation attenuates TCR signaling, leading to T cell inhibition. To promote this event, a bispecific fusion protein comprising a mutant mouse CD80 (CD80w88a) and lymphocyte activation antigen-3 was engineered to concurrently engage CTLA-4 and crosslink it to the TCR. Crosslinking is expected to be attained via ligation of CTLA-4 first to MHCII and then indirectly to the TCR, generating a CTLA-4-MHCII-TCR tri-molecular complex that forms between T cells and antigenpresenting cells during T cell activation. Treating T cells with this bispecific fusion protein in vitro inhibited T cell activation, induced production of IL-10 and TGF-β, and attenuated AKT and mTOR signaling. Intriguingly, the bispecific fusion protein also directed early T cell differentiation into Foxp3 positive regulatory T cells (Tregs) in a process that was dependent on the endogenous production of TGF-β. Treatment of non-obese diabetic (NOD) female mice between 4-13 weeks of age with the bispecific protein significantly delayed the onset of disease or protected animals from developing autoimmune diabetes. Thus, bispecific fusion proteins that engage CTLA-4 and co-ligate it to the TCR during the early phase of T cell activation can negatively regulate the T cell response at multiple levels. Bispecific biologics with such dual functions may therefore represent a novel class of therapeutics for immune modulation. These findings presented here also reveal a potential new role for CTLA-4 in Treg differentiation. Yunxiang Zhu et al., J Clin Cell Immunol 2013, 4:5 http://dx.doi.org/10.4172/2155-9899.S1.013Objective: To analyze the risk factors affecting postoperative infectious complications in HIV-infected patients and explore the rational use of perioperative antibiotics. Methods: Retrospective analysis of 308 HIV-infected patients (male 272, female 36) who were operated at the Shanghai Public Health Clinical Center from Nov 2008 to Apr 2012. The patients were divided into postoperative infection and non-infection groups. Age and clinical variables were compared. The correlation between the method of surgical incision, surgical site infection (SSI) and postoperative sepsis were analyzed. Prophylactic antibiotics were used for patients with type I and II surgical incisions for no longer than 2 days. Patients with type III surgical incisions were administered antibiotics until infection was controlled. Antiretroviral therapy (ART) were used before operation for patients whose preoperative CD4 counts were <350 cells/μL,For those patients whose preoperative CD4 counts were <200 cells/μL, Sulfamethoxazole and fluconazole were administered preoperatively as a prophylaxis against Pneumocystis carinii pneumonia and fungal infection. Results: 196 patients developed postoperative infectious complications with 7 mortalities. Preoperative CD4 counts, ratio of CD4/ CD8 cells, hemoglobin level, and postoperative CD4 counts, hemoglobin and albumin levels were risk factors of perioperative infection in HIV-infected patients. Patients with a preoperative CD4 count < 200cell/μl, anemia, or a postoperative CD4 count < 200cell/μl and albumin levels < 35g/L correlated with a higher rate of postoperative infection. There was a significant correlation between SSI and the type of surgical incision. The rate of SSI in patients with type I surgical incision was 2% and the patients with type II surgical incision was 38%. All the patients who received type III surgical incisions developed SSI. Patients with SSI were more likely to develop postoperative sepsis. Conclusions: HIV-infected patients are more likely to develop postoperative infectious complications. Rational use of antibiotics in HIV-infected patients could help to reduce the rate of postoperative infectious complications in these patients. Baochi Liu et al., J Clin Cell Immunol 2013, 4:5 http://dx.doi.org/10.4172/2155-9899.S1.013N Diabetes after Transplantation (NODAT) is a serious metabolic complication after kidney transplantation, associated with poorer patient and graft survival. This study aimed to determine the incidence of NODAT and to identify risk factors for development of NODAT among kidney transplant recipients. Materials and Methods: This is an observational study of 401 patients who were transplanted between Jan 2000Jan 2008 at Mahavir hospital. NODAT was determined using criteria as per American Diabetes Association/ WHO guidelines. Logistic regression analyses were performed to identify predictors of NODAT. Results: Among 401 patients included in the study NODAT was observed in 59 patients (14.7%) after a mean follow-up time of 58.7 months. The mean age of the patients was 39.7±9.1 years and 69.1% were men and 30.1% were women; 26.9% received cyclosporine (CsA) and 73.1% tacrolimus (Tac). The incidence of NODAT was 2.74%, 4.98% and 6.98% at 1, 3, and 5 years following transplantation. In CsA-treated patients, the NODAT was 5.5% and 8.4% at 1and 5year post-transplantation, while in Tac-treated patients, it was 15.6% and 19.8%. Risk factors of NODAT were older age, use of CNIs, acute rejections and viral infections like HCV and CMV. Conclusion: We conclude that high incidences of NODAT are associated with the type of initial maintenance immunosuppression, acute rejections, obesity, hepatitis C infection and family history of diabetes. It is one of factor of graft failure and mortality. Sailaja kesiraju et al., J Clin Cell Immunol 2013, 4:5 http://dx.doi.org/10.4172/2155-9899.S1.013Background: Human Leucocyte Antigen-G (HLA-G) molecules are involved in the inhibition of cell-mediated immune responses and could promote the propagation of HIV-1 infection across the placental interface thus increasing the risk of vertical transmission. Therefore, the objective of this study was to assess whether the Major Histocompatibility Complex (MHC) - coded molecule HLA-G inhibits Natural Killer (NK) cell activity thereby, assisting viral penetration across the placental barrier in HIV-1 positive pregnant women. nStudy Design & Methods: Natural Killer (CD56+) cell activity and placental HLA-G1 expression was assessed using immunohistochemistry and real-time polymerase chain reaction (RT-PCR) techniques, respectively. Studies were performed on a total of fifty five placental samples obtained from HIV-1 infected mothers at birth. nResults: Low numbers of NK cells increased risk of vertical transmission [OR = 3.424 (95%CI 0.65-17.89)]. The risk of babies becoming infected increased by 1.3 with every 1 unit increase in HLA-G1 expression. A positive correlation was observed between mothers log viral load and transmission of infection to the baby (p = 0.047; 95%CI 1.029-11.499). nConclusion: Low NK cell activity at the placental interface increased the risk of vertical transmission. Maternal viral load remained a strong predictor of viral transmission.C molecules are targeted for immune intervention in inflammatory and autoimmune diseases. Among the costimulatory molecules, CD2-CD58 molecular pairs provide the adhesion between T cell and antigen presenting cell for signaling in the very early stage of immune response. The protein-protein interaction (PPI) between CD2 and CD58 (CD48 in rodents) helps enhance T cell-antigen-presenting cells (APC) adhesion and thus promotes T-cell activation. Studies related to structure of proteins indicated that PPI surfaces have “hot-spots” that are hydrophobic in nature and PPI can be modulated by targeting small molecules or peptides. Our strategy is to use conformationally constrained peptides to inhibit proteinprotein interactions in the key regions of the CD2-CD58 interactions. The result of blocking CD2-CD58 interaction will lead to suppression of T-cell activation and is clinically important for the treatment of autoimmune diseases. We have designed cyclic peptides and peptidomimetics from the surface epitopes of CD2 protein to inhibit CD2-CD58/CD48 interaction ultimately resulting in immunomodulation. The cell adhesion inhibition activity of the designed peptides (compound 6 and 7) were studied by cell adhesion assay. Compounds 6 and 7 inhibit the cell adhesion with an IC50 value of 6 and 11 nM respectively. Antibody binding inhibition assay indicated that these compounds bind to cells expressing CD58/CD48 proteins. Compounds were evaluated for their ability to modulate the immune response in collagen induced arthritis model (CIA). Results suggested that both the compounds were able to suppress the progression of arthritis in CIA model and were able to suppress T cell response in transgenic animal model. Seetharama Satyanarayanajois et al., J Clin Cell Immunol 2013, 4:5 http://dx.doi.org/10.4172/2155-9899.S1.013

Abstract 5420: Normal mammary epithelial mitochondria introduced into human breast cancer cells results in inhibition of proliferation and increased drug sensitivity of the MCF-7 breast cancer cell line.

Warburg in the 1920s postulated that mitochondrial dysfunction alters metabolism in cancer. He observed in cancer cells aerobic glycolysis, which is the conversion of glucose to lactic acid in the presence of oxygen, the so called “Warburg effect” [1]. Mitochondrial dysfunction of cancer cells includes increased aerobic glycolysis, elevated levels of ROS, decreased apoptosis, and resistance to chemotherapeutic agents. We reported on the ultrastructural observations of mitochondria in human breast carcinoma cells [2]. There were three distinct groups: (1) mitochondria absent, (2) mitochondria present, (3) mitochondria present but sparse and abnormal. Tumors in the absent group were more anaplastic, aggressive, and resistant to treatment. We hypothesized that the introduction of normal mitochondria into cancer cells might restore mitochondrial function and inhibit cancer cell growth, and reverse chemoresistance. First we tested if mitochondria of normal mammary epithelial MCF-12A cells could enter into cultured human cancer cells. Then we determined if introducing normal mitochondria into cancer cells would inhibit proliferation. Finally we determined if addition of normal mitochondria increases the sensitivity of human breast cancer MCF-7 cells to chemotherapy. We found JC-1 stained mitochondria from normal mammary epithelial MCF-12A cells can enter into the cancer cell lines MCF-7, MDA-MB-231, and NCI/ADR-Res, but cannot enter normal MCF-12 A cells. The normal mitochondria from normal MCF-12 A cells suppressed the proliferation of MCF-7 and NCI/ADR-Res cells in a dose dependent pattern, but did not affect the proliferation of normal MCF-12A cells. The normal mitochondria from normal MCF-12A cells increased the sensitivity of human breast cancer MCF-7 cells to doxorubicin, Abraxane, and carboplatin. In conclusion the introduction of normal mammary mitochondria into human cancer cells inhibits cancer cell proliferation and increases the sensitivity of the MCF-7 human breast cancer cells to doxorubicin, Abraxane, and carboplatin. This phenomenon has never been reported, and the results support the role of mitochondrial dysfunction in cancer and suggest the possible use of targeted mitochondria as an adjunct to cancer therapeutics. Citation Format: Robert L. Elliott, Xian P. Jian, Jonathan F. Head. Normal mammary epithelial mitochondria introduced into human breast cancer cells results in inhibition of proliferation and increased drug sensitivity of the MCF-7 breast cancer cell line. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5420. doi:10.1158/1538-7445.AM2013-5420

Abstract 1507: Iron upregulates HLA-G mRNA expression of the human MCF-7 breast cancer cell line.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DCnnHLA-G is a nonclassical MHC Class I molecule that protects the fetus from the maternal immune system, and it also plays a role in immune evasion of cancer. Previously we have shown that iron can suppress the cytolytic function of natural killer cells to human breast cancer cell lines. We have found that breast cancer cells over express HLA-G mRNA and proteins in both cell lines and breast carcinomas. In order to determine if iron concentration affects HLA-G expression, we cultured the human breast cancer cell line MCF-7 in media with 100 mM, 400 mM, and 1600 mM of iron for 24 hours. We used RT-PCR to examine HLA-G mRNA expression of the MCF-7 cells. The PCR primers were designed so that they could detect all alternatively spliced HLA-G mRNA isoforms, 1000 bp (HLA-G1 and HLA-G5), 600 bp (HLA-G2 and HLA-G4), and 300 bp (HLA-G3). RT-PCR showed that the MCF-7 cells cultured with control medium only had one band of 300 bp mRNA expression. However, the MCF-7 cells cultured in medium containing 100 mM, 400 mM, and 1600 mM of iron had three bands of 300 bp, 600 bp, and 1000 bp HLA-G mRNA expression. Moreover, the intensity of the 300 bp band from the iron-treated MCF-7 cells is significantly higher than the cells cultured with control medium. In summary, iron upregulates the HLA-G mRNA expression of MCF-7 cells, and this probably contributes to cancer immunosuppression and to the cytolytic dysfunction of natural killer cells.nnCitation Format: Jonathan F. Head, Xian P. Jiang, Robert L. Elliott. Iron upregulates HLA-G mRNA expression of the human MCF-7 breast cancer cell line. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1507. doi:10.1158/1538-7445.AM2013-1507