STAT3 activation by the ErbB2 receptor and development of peptide-based STAT3 inhibitors

Stats (s&barbelow;ignal t&barbelow;ransducer and a&barbelow;ctivator of t&barbelow;ranscription) are latent transcription factors that translocate from the cytoplasm to nucleus. Constitutive activation of Stat3α by upstream oncoproteins and receptor tyrosine kinases has been found in many human tumors and tumor-derived cell lines and it is often correlated with the activation of ErbB-2. In order to explore the involvement of ErbB-2 in the activation of Stat3 and the mechanisms underlying this event, an erbB-2 point mutant was used as a model of a constitutively activated receptor. Phenylalanine mutations (Y-F) were made in the receptor's autophosphorylation sites and their ability to activate Stat3α was evaluated. Our results suggest that Stat3α and Janus tyrosine kinase 2 associates with ErbB-2 prior to tyrosine phosphorylation of the receptor and that full activation of Stat3α by ErbB-2 requires the participation of other non-receptor tyrosine kinases. Both Src and Jak2 kinases contribute to the activation of Stat3α while only Src binds to ErbB-2 only when the receptor is tyrosine phosphorylated. Our results also suggest that tyrosine 1139 may be important for Src SH2 domain association since a mutant lacking this tyrosine reduces the ability of the Src SH2 domain to bind to ErbB-2 and significantly decreases its ability to activate Stat3α. ^ In order to disrupt aberrant STAT3α activation which contributes to tumorigenesis, we sought small molecules which can specifically bind to the STAT3 SH2 domain, thereby abolishing its ability of being recruited into receptors, and also blocking the dimer formation required for STAT3α activation. A phosphopeptide derived from gp130 was found to have a high affinity to STAT3 SH2 domain, and we decided to use this peptide as the base for further modifications. A series of peptide based compounds were designed and tested using electrophoretic mobility shift assay and fluorescence polarization assay to evaluate their affinity to the STAT3 SH2 domain. Two promising compounds, DRIV-73C and BisPOM, were used for blocking STAT3α activity in cell culture. Either can successfully impair STAT3α activation induced by IL-6 stimulation in HepG2 cells. BisPOM proved to be the more effective in blocking STAT3α tyrosine phosphorylation in induced cells and tumor cell lines, and was the more potent in inhibiting STAT3 dependent cell growth. ^

{\it In vivo\/} induction of cytotoxic T lymphocytes against human immunodeficiency virus type 1 by synthetic viral peptides

Cytotoxic T lymphocytes (CTLs) play an important role in the suppression of initial viremia after acute infection with the human immunodeficiency virus (HIV), the causative agent of acquired immune deficiency syndrome (AIDS). Most HIV-infected individuals attain a high titer of anti-HIV antibodies within weeks of infection; however this antibody-mediated immune response appears not to be protective. In addition, anti-HIV antibodies can be detrimental to the immune response to HIV through enhancement of infection and participating in autoimmune reactions as a result of HIV protein mimicry of self antigens. Thus induction and maintenance of a strong HIV-specific CTL immune response in the absence of anti-HIV antibodies has been proposed to be the most effective means of controlling of HIV infection. Immunization with synthetic peptides representing HIV-specific CTL epitopes provides a way to induce specific CTL responses, while avoiding stimulation of anti-HIV antibody. This dissertation examines the capacity of synthetic peptides from the V3 loop region of the gp120 envelope protein from several different strain of HIV-1 to induce HIV-specific, MHC-restricted CD8$\sp+$ CTL response in vivo in a mouse model. Seven synthetic peptides representative of sequences found throughout North America, Europe, and Central Africa have been shown to prime CTLs in vivo. In the case of the MN strain of HIV-1, a 13 amino acid sequence defining the epitope is most efficient for optimal induction of specific CTL, whereas eight to nine amino acid sequences that could define the epitope were not immunogenic. In addition, synthesis of peptides with specific amino acid substitutions that are important for either MHC binding or T cell receptor recognition resulted in peptides that exhibited increased immunogenicity and induced CTLs that displayed altered specificity. V3 loop peptides from HIV-1 MN, SC, and Z321 induced a CTL population that was broadly cross-reactive against strains of HIV-1 found throughout the world. This research confirms the potential efficacy of using synthetic peptides for in vivo immunization to induce HIV-specific CTL-mediated responses and provides a basis for further research into development of synthetic peptide-based vaccines. ^

Immune responses against major histocompatibility complex and interleukin-2 gene transfected K1735 murine melanoma: Potential vaccines for the immunotherapy of cancer

Tumor specific immunity is mediated by cytotoxic T lymphocytes (CTL) that recognize peptide antigen (Ag) in the context of major histocompatibility complex (MHC) class I molecules and by helper T (Th) lymphocytes that recognize peptide Ag in the context of MHC class II molecules. The purpose of this study is (1) to induce or augment the immunogenicity of nonimmunogenic or weakly immunogenic tumors by genetic modification of tumor cells, and (2) to use these genetically altered cells in cancer immunotherapy. To study this, I transfected a highly tumorigenic murine melanoma cell line (K1735) that did not express constitutively either MHC class I or II molecules with syngeneic cloned MHC class I and/or class II genes, and then determined the tumorigenicity of transfected cells in normal C3H mice. K1735 transfectants expressing either $\rm K\sp{k}$ or $\rm A\sp{k}$ molecules alone produced tumors in normal C3H mice, whereas most transfectants that expressed both molecules were rejected in normal C3H mice but produced tumors in nude mice. The rejection of K1735 transfectants expressing $\rm K\sp{k}$ and $\rm A\sp{k}$ Ag in normal C3H mice required both $\rm CD4\sp+$ and $\rm CD8\sp+$ T cells. Interestingly, the $\rm A\sp{k}$ requirement can be substituted by IL-2 because transfection of $\rm K\sp{k}$-positive/A$\sp{\rm k}$-negative K1735 cells with the IL-2 gene also resulted in abrogation of tumorigenicity in normal C3H mice but not in nude mice. In addition, 1735 $(\rm I\sp+II\sp+)$ transfected cells can function as antigen presenting cells (APC) since they could process and present native hen egg lysozyme (HEL) to HEL specific T cell hybridomas. Furthermore, the transplantation immunity induced by K1735 transfectants expressing both $\rm K\sp{k}$ and $\rm A\sp{k}$ molecules completely cross-protected mice against challenge with $\rm K\sp{k}$-positive transfectants but weakly protected them against challenge with parental K1735 cells or $\rm A\sp{k}$-positive transfectants. Finally, I demonstrated that MHC $(\rm I\sp+II\sp+)$ or $\rm K\sp{k}$-positive/IL-2-positive cells can function as anti-cancer vaccines since they can abrogate the growth of established tumors and metastasis.^ In summary, my results indicate that expression of either MHC class I or II molecule alone is insufficient to cause the rejection of K1735 melanoma in syngeneic hosts and that both molecules are necessary. In addition, my data suggest that the failure of $\rm K\sp{k}$-positive K1735 cells to induce a primary tumor-rejection response in normal C3H mice may be due to their inability to induce the helper arm of the anti-tumor immune response. Finally, the ability of MHC $(\rm I\sp+II\sp+)$ or $\rm K\sp{k}$-positive/IL-2-positive cells to prevent growth of established tumors or metastasis suggests that these cell lines can serve as potential vaccines for the immunotherapy of cancer. (Abstract shortened by UMI.) ^

Peptide nucleic acids: Mechanism of tight binding and application in artificial nuclease

Peptide nucleic acids (PNA) are mimics of nucleic acids with a peptidic backbone. Duplexes and triplexes formed between PNA and DNA or RNA possess remarkable thermal stability, they are resistant to nuclease cleavage and can better discriminate mismatches. Understanding the mechanism for the tight binding between PNA and oligonucleotides is important for the design and development of better PNA-based drugs.^ We have performed molecular dynamics (MD) simulations of 8-mer PNA/DNA duplex and two analogous duplexes with chiral modification of PNA strand (D- or L-Alanine modification). MD simulations were performed with explicit water and Na$\sp{+}$ counter ions. The 1.5-ns simulations were carried out with AMBER using periodic boundary and particle mesh Ewald summation. The point charges for PNA monomers were derived from fitting electrostatic potentials, obtained from ab initio calculation, to atomic centers using RESP. Derived charges reveal significantly altered charge distribution on the PNA bases and predict the Watson-Crick H-bonds involving PNA to be stronger. Results from NMR studies investigating H-bond interactions between DNA-DNA and DNA-PNA base pairs in non-polar environment are consistent with this prediction. MD simulations demonstrated that the PNA strand is more flexible than the DNA strand in the same duplex. That this flexibility might be important for the duplex stability is tested by introducing modification into the PNA backbones. Results from MD simulation revealed dramatically altered structures for the modified PNA-DNA duplexes. Consistent with previous NMR results, we also found no intrachain hydrogen bonds between O7$\sp\prime$ and N1$\sp\prime$ of the neighboring residues in our MD study. Our study reveals that in addition to the lack of charge repulsion, stronger Watson-Crick hydrogen bonds together with flexible backbone are important factors for the enhanced stability of the PNA-DNA duplex.^ In a related study, we have developed an application of Gly-Gly-His-(Gly)$\sb3$-PNA conjugate as an artificial nuclease. We were able to demonstrate cleavage of single stranded DNA at a single site upon Ni(II) binding to Gly-Gly-His tripeptide and activation of nuclease with monoperoxyphthalic acid. ^

Future pandemic influenza control (FPIC) related strategic management plan based on the 2009 H1N1 pandemic influenza review of health policy decisions, their practice and implications

This cross-sectional study is based on the qualitative and quantitative research design to review health policy decisions, their practice and implications during 2009 H1N1 influenza pandemic in the United States and globally. The “Future Pandemic Influenza Control (FPIC) related Strategic Management Plan” was developed based on the incorporation of the “National Strategy for Pandemic Influenza (2005)” for the United States from the U.S. Homeland Security Council and “The Canadian Pandemic Influenza Plan for the Health Sector (2006)” from the Canadian Pandemic Influenza Committee for use by the public health agencies in the United States as well as globally. The “global influenza experts’ survey” was primarily designed and administered via email through the “Survey Monkey” system to the 2009 H1N1 influenza pandemic experts as the study respondents. The effectiveness of this plan was confirmed and the approach of the study questionnaire was validated to be convenient and the excellent quality of the questions provided an efficient opportunity to the study respondents to evaluate the effectiveness of predefined strategies/interventions for future pandemic influenza control.^ The quantitative analysis of the responses to the Likert-scale based questions in the survey about predefined strategies/interventions, addressing five strategic issues to control future pandemic influenza. The effectiveness of strategies defined as pertinent interventions in this plan was evaluated by targeting five strategic issues regarding pandemic influenza control. For the first strategic issue pertaining influenza prevention and pre pandemic planning; the confirmed effectiveness (agreement) for strategy (1a) 87.5%, strategy (1b) 91.7% and strategy (1c) 83.3%. The assessment of the priority level for strategies to address the strategic issue no. (1); (1b (High Priority) > 1a (Medium Priority) > 1c (Low Priority) based on the available resources of the developing and developed countries. For the second Strategic Issue encompassing the preparedness and communication regarding pandemic influenza control; the confirmed effectiveness (agreement) for the strategy (2a) 95.6%, strategy (2b) 82.6%, strategy (2c) 91.3% and Strategy (2d) 87.0%. The assessment of the priority level for these strategies to address the strategic issue no. (2); (2a (highest priority) > 2c (high priority) >2d (medium priority) > 2b (low priority). For the third strategic issue encompassing the surveillance and detection of pandemic influenza; the confirmed effectiveness (agreement) for the strategy (3a) 90.9% and strategy (3b) 77.3%. The assessment of the priority level for theses strategies to address the strategic Issue No. (3) (3a (high priority) > 3b (medium/low priority). For the fourth strategic issue pertaining the response and containment of pandemic influenza; the confirmed effectiveness (agreement) for the strategy (4a) 63.6%, strategy (4b) 81.8%, strategy (4c) 86.3%, and strategy (4d) 86.4%. The assessment of the priority level for these strategies to address the strategic issue no. (4); (4d (highest priority) > 4c (high priority) > 4b (medium priority) > 4a (low priority). The fifth strategic issue about recovery from influenza and post pandemic planning; the confirmed effectiveness (agreement) for the strategy (5a) 68.2%, strategy (5b) 36.3% and strategy (5c) 40.9%. The assessment of the priority level for strategies to address the strategic issue no. (5); (5a (high priority) > 5c (medium priority) > 5b (low priority).^ The qualitative analysis of responses to the open-ended questions in the study questionnaire was performed by means of thematic content analysis. The following recurrent or common “themes” were determined for the future implementation of various predefined strategies to address five strategic issues from the “FPIC related Strategic Management Plan” to control future influenza pandemics. (1) Pre Pandemic Influenza Prevention, (2) Seasonal Influenza Control, (3) Cost Effectiveness of Non Pharmaceutical Interventions (NPI), (4) Raising Global Public Awareness, (5) Global Influenza Vaccination Campaigns, (6)Priority for High Risk Population, (7) Prompt Accessibility and Distribution of Influenza Vaccines and Antiviral Drugs, (8) The Vital Role of Private Sector, (9) School Based Influenza Containment, (10) Efficient Global Risk Communication, (11) Global Research Collaboration, (12) The Critical Role of Global Public Health Organizations, (13) Global Syndromic Surveillance and Surge Capacity and (14) Post Pandemic Recovery and Lessons Learned. The future implementation of these strategies with confirmed effectiveness to primarily “reduce the overall response time’ in the process of ‘early detection’, ‘strategies (interventions) formulation’ and their ‘implementation’ to eventually ensure the following health outcomes: (a) reduced influenza transmission, (b) prompt and effective influenza treatment and control, (c) reduced influenza related morbidity and mortality.^

THE CYTOPLASMIC TAIL OF MHC CLASS I MOLECULES PLAYS A CRITICAL ROLE IN DENDRITIC CELL-INDUCED T CELL IMMUNITY

The presentation of MHC class I (MHC-I)/peptide complexes by dendritic cells (DCs) is critical for the maintenance of central tolerance to self and for the regulation of cytotoxic T lymphocytes (CTL)-mediated adaptive immune responses against pathogens and cancer cells. Interestingly, several findings have suggested that the cytoplasmic tail of MHC class I plays a functional role in the regulation of CTL immune responses. For example, our previous studies demonstrated that exon 7-deleted MHC-I molecules not only showed extended DC cell surface half-lives but also induced significantly increased CTL responses to viral challange invivo. Although exon 7-deleted variant of MHC-I does not occur naturally in humans, the animal studies prompted us to examine whether exon 7-deleted MHC-I molecules could generate augmented CTL responses in a therapeutic DC-based vaccine setting. To examine the stimulatory capacity of exon 7-deleted MHC-I molecules, we generated a lentivirus-mediated gene transfer system to induce the expression of different MHC-I cytoplasmic tail isoforms in both mouse and human DCs. These DCs were then used as vaccines in a melanoma mouse tumor model and in a human invitro co-culture system. In this thesis, we show that DCs expressing exon 7-deleted MHC-I molecules, stimulated remarkably higher levels of T-cell cytokine production and significantly increased the proliferation of meanoma-specific (Pmel-1) T cells compared with DCs expressing wild type MHC-I. We also demonstrate that, in combination with adoptive transfer of Pmel-1 T-cell, DCs expressing exon 7-deleted Db molecules induced greater anti-tumor responses against established B16 melanoma tumors, significantly extending mouse survival as compared to DCs expressing wild-type Db molecules. Moreover, we also observed that human DCs expressing exon 7-deleted HLA-A2 molecules showed similarly augmented CTL stimulatory ability. Mechanistic studies suggest that exon 7-deleted MHC-I molecules showed impaired lateral membrane movement and extended cell surface half-lives within the DC/T-cell interface, leading to increased spatial availability of MHC-I/peptide complexes for recognition by CD8+ T cells. Collectively, these results suggesr that targeting exon 7 within the cytoplasmic tail of MHC-I molecules in DC vaccines has the potential to enhance CD8+ T cell stimulatory capacity and improve clinical outcomes in patients with cancer or viral infections.