Thesis (M.A.B.S.)--Multnomah Graduate School of Ministry, 1987. / Includes bibliographical references (leaves 66-70).

Reconstructive flaps are a mainstay form of treatment for anatomical defects in plastic surgery, and despite extensive progress in the areas of flap anatomy and design, the mechanism of flap healing and the factors that regulate this process are poorly understood. This thesis investigates the regulation of flap healing, and tests the hypothesis that the introduction of genes for angiogenic growth factors can be used to augment the vascularisation and wound healing of ischaemic flaps. Using a modified McFarlane ischaemic skin flap model in Sprague Dawley rats, endogenous angiogenic regulatory factors that included the VEGF and angiopoietin families and their receptors were investigated. Twelve specific quantitative real-time PCR assays documented a general up-regulation of angiogenic genes and their receptors with time following flap elevation. There was not a readily identifiable “master regulator”. Angiogenic protein levels were more variable with a decrease VEGF-A and TNF-α levels along the flap. Debridement studies of the necrotic distal flaps demonstrated for the first time that VEGF-A164 and TNF-α protein levels stabilised, while angiogenic genes of VEGF-A164, VEGF-A120, angiopoietins and their receptors were down-regulated and VEGF-B186 and HIF-1α mRNA increased, compared to non-debrided flaps. Leucocyte proteolysis in devitalised tissue is discussed as a possible mechanism for reduced angiogenic proteins levels in ischaemic flaps. The impact of angiogenic gene therapy using adenoviral vectors in the flap model revealed for the first time that recombinant adenoviruses containing the VEGF-B186 transgene could significantly augment neovascularisation and improve flap survival. This neovascularisation correlated with up-regulation of the expression of multiple endogenous angiogenic genes that included VEGF-A164, the angiopoietins and their receptors. Erythematous plaques were documented as a side effect of Ad VEGF-A165 and Ad VEGF-B186 treatment of rat skin, although in the latter treatment they were very mild. Weals induced by the presence of VEGF-A165 transgene were associated with a marked acute inflammatory cell infiltrate and oedema consistent with the increased vascular permeability effects of VEGF-A165. Ad VEGF-A165 plus Ad ANG-1* induced weals were less prominent with reduced oedema highlighting the stabilising effect of Ad ANG-1* on vascular permeability.

<p>The Signalosome is a term used to define a putative signalling complex, which assembles near the plasma membrane in response to external signals received at cell surface receptors and then migrates towards downstream effectors. It is proposed to regulate the level of intracellular Ca2+ and subsequent downstream signalling events. To date it has been defined to consist of BTK, BLNK, BCAP, VAV, PLCγ2 and PI3K1-4 in B-Cells.</p><p>This work entailed initiating a new proteomic approach to investigate the nature and extent of Bruton’s tyrosine kinase, Btk, involvement in the signalosome – inherently, the aim was to study multiple interactions of Btk with other molecules. By transfecting host cells with a Btk gene-transfer plasmid, virus particles were produced that were used to up-regulate and analyse the expression of Btk in three haematopoietic cell lines: B-cells, Pre-B-cells and a myeloid cancer cell. The construction of a new gene-transfer vector was successfully carried out by plasmid sub-cloning and it was subsequently found to effectively transfect the host cells and produce virus particles. The recombinant virus particles were employed with success in transducing three haematopoietic cell lines and with immunopurification and subsequent gel separation protein signalosome complexes were obtained ready for analysis by mass spectrometrical fingerprinting (to be carried out as a joint effort in Mount Sinai Hospital in Toronto, Canada).</p>

The study analyzes the ethical implications of human gene transfer technology for enhancement. Although human gene transfer technology is widely accepted on therapeutic grounds the non-therapeutic use of gene transfer technology remains to be a gray zone for moral deliberation. The present discussion addresses several ethical issues concerning the impacts of human gene transfer technology on individuals, the society, and future people. Accordingly, the study examines major ethical issues concerning the use of human gene transfer technology in general and genetic enhancement in particular, and reliability of the putative demarcation between therapy and enhancement, and further proposes ethical guidelines for non-therapeutic application of human gene transfer technology. A special attention is given to three major ethical issues, such as our obligation to future generations, problems concerning justice, fairness, and equality, and the problem of uncertainty.

The Signalosome is a term used to define a putative signalling complex, which assembles near the plasma membrane in response to external signals received at cell surface receptors and then migrates towards downstream effectors. It is proposed to regulate the level of intracellular Ca2+ and subsequent downstream signalling events. To date it has been defined to consist of BTK, BLNK, BCAP, VAV, PLCγ2 and PI3K1-4 in B-Cells. This work entailed initiating a new proteomic approach to investigate the nature and extent of Bruton’s tyrosine kinase, Btk, involvement in the signalosome – inherently, the aim was to study multiple interactions of Btk with other molecules. By transfecting host cells with a Btk gene-transfer plasmid, virus particles were produced that were used to up-regulate and analyse the expression of Btk in three haematopoietic cell lines: B-cells, Pre-B-cells and a myeloid cancer cell. The construction of a new gene-transfer vector was successfully carried out by plasmid sub-cloning and it was subsequently found to effectively transfect the host cells and produce virus particles. The recombinant virus particles were employed with success in transducing three haematopoietic cell lines and with immunopurification and subsequent gel separation protein signalosome complexes were obtained ready for analysis by mass spectrometrical fingerprinting (to be carried out as a joint effort in Mount Sinai Hospital in Toronto, Canada).

The success of adenoviral mediated airway gene therapy is hindered by host immune responses against adenoviral vectors. Helper-dependent adenoviral vectors (HD-Ad) are devoid of viral coding sequences and have an improved safety profile compared to earlier generation adenoviral vectors. However, intranasal delivery of HD-Ad vectors potentiates a pulmonary adaptive immune response, described in chapter 2, which is a barrier to gene therapy. One of the ways to reduce the immunogenicity of HD-Ad vectors is to increase the efficiency of HD-Ad mediated gene transfer to the airways, which would lessen the immunogen availability, limiting immune response against HD-Ad vectors. In chapter 3, a viral formulation strategy using Nacystelyn and DEAE-Dextran to substantially increase the efficacy of adenoviral mediated gene transfer to the airways is described. To further reduce the immune response to HD-Ad vectors, I have developed two novel strategies to induce vector-specific tolerance. The first strategy, described in chapter 4, involves the use of dendritic cells (DCs) differentiated in presence of IL-10, which are refractory to HD-Ad induced maturation and instead prime generation of regulatory T cells which suppress HD-Ad induced T cell proliferation. Delivery of these DCs pulsed with HD-Ad vectors to mice results in induction of immunological tolerance along with sustained gene expression following multiple rounds of HD-Ad readministrations. The second strategy, described in chapter 5, involves delivery of apoptotic DCs followed by delivery of antigen towards which tolerance needs to be generated. Apoptotic DCs are readily taken up by viable DCs, which suppresses DC maturation and induces TGF-β1 secretion, driving generation of regulatory T cells towards the delivered antigen. This strategy has shown remarkable success in achieving tolerance towards ovalbumin. Therefore, these strategies can be used to induce immunological tolerance towards gene therapy vectors which will likely allow for sustained and long term therapeutic transgene expression.

The success of adenoviral mediated airway gene therapy is hindered by host immune responses against adenoviral vectors. Helper-dependent adenoviral vectors (HD-Ad) are devoid of viral coding sequences and have an improved safety profile compared to earlier generation adenoviral vectors. However, intranasal delivery of HD-Ad vectors potentiates a pulmonary adaptive immune response, described in chapter 2, which is a barrier to gene therapy. One of the ways to reduce the immunogenicity of HD-Ad vectors is to increase the efficiency of HD-Ad mediated gene transfer to the airways, which would lessen the immunogen availability, limiting immune response against HD-Ad vectors. In chapter 3, a viral formulation strategy using Nacystelyn and DEAE-Dextran to substantially increase the efficacy of adenoviral mediated gene transfer to the airways is described. To further reduce the immune response to HD-Ad vectors, I have developed two novel strategies to induce vector-specific tolerance. The first strategy, described in chapter 4, involves the use of dendritic cells (DCs) differentiated in presence of IL-10, which are refractory to HD-Ad induced maturation and instead prime generation of regulatory T cells which suppress HD-Ad induced T cell proliferation. Delivery of these DCs pulsed with HD-Ad vectors to mice results in induction of immunological tolerance along with sustained gene expression following multiple rounds of HD-Ad readministrations. The second strategy, described in chapter 5, involves delivery of apoptotic DCs followed by delivery of antigen towards which tolerance needs to be generated. Apoptotic DCs are readily taken up by viable DCs, which suppresses DC maturation and induces TGF-β1 secretion, driving generation of regulatory T cells towards the delivered antigen. This strategy has shown remarkable success in achieving tolerance towards ovalbumin. Therefore, these strategies can be used to induce immunological tolerance towards gene therapy vectors which will likely allow for sustained and long term therapeutic transgene expression.

The efficiency of retrovirus-mediated gene transfer can be dramatically enhanced by inducing flocculation of viruses. Addition of oppositely charged polymers to virus stocks resulted in the formation of virus-polymer complexes. The complexes specifically incorporated virus particles and only few other proteins, were not cytotoxic, did not reduce the stability of the viruses, and were large enough to sediment, delivering the viruses to the cells more rapidly than by simple diffusion. Increases in the rate of transport of viruses correlated with increases in the rate of transduction, as the polymers did not affect the efficiency of post-binding steps of transduction. The formation of virus-polymer complexes also permitted concentrating viruses and purifying the stocks from inhibitors of transduction. Pelleting of the complexes followed by resuspension of the pellet in a reduced volume of fresh cell culture medium resulted in substantial enhancement of transduction. Purified virus stocks could be used in smaller quantities than unprocessed stocks to achieve a given level of gene transfer and reduced uncertainties about the relationship between the amount of virus used and the number of genes transferred. When using high concentrations of purified viruses, the efficiency of gene transfer was dependent on the number of envelope proteins displayed on the surface of each virus particle. Viruses with a low number of envelope proteins transduced cells more efficiently than did viruses with a high number of envelope proteins, and allowed more integrations of the transgene per target cell. In contrast, when the number of envelope proteins per virus particle was high, transduction appeared to be limited by a reduction in availability of functional receptors for viruses pseudotyped with the same envelope. Taken together, this novel method for processing retrovirus stocks and a better understanding of major limitations of transduction should simplify efforts to predict the outcome of retrovirus transduction protocols and should help to increase the likelihood that human gene therapy protocols will succeed.

Despite the development of novel target therapy drugs in recent years, metastatic cancer remains refractory to current cancer therapies and accounts for the majority of cancer mortalities worldwide. Metastasis consists of multiple steps including angiogenesis, extravasion, escape from immune surveillance, adhesion, and clonal expansion in different organs that a systemic therapy is required for effective control of metastasis. The pro-inflammatory nuclear factor kappa B (NF£eB) pathway plays an important role during each of these metastatic events and constitutes an excellent target for metastasis control. Stress hormone pro-opiomelanocortin (POMC) and its derived neuropeptides including corticotrophin (ACTH), £\-, £]-, and £^-melanocyte¡Vstimulating hormone (£\-, £]-, and £^-MSH), £]-endorphin are potent inhibitors of NF£eB pathway. Other than the central regulation of stress response and energy homeostasis, POMC also regulates the skin pigmentation, inflammatory processes, and immune reactions in the peripheral system. Since adenovirus¡Vmediated POMC gene delivery leads to hepatic POMC expression, it seems plausible that POMC gene therapy may elicit systemic production of anti-inflammatory POMC-derived peptides and hold promises for control of primary and metastatic cancers. In B16-F10 melanoma models, POMC gene delivery elevated the circulating ACTH levels for more than 8 weeks and suppressed the growth of established melanoma, thereby prolonging the life span of tumor-bearing mice. Moreover, combination of POMC therapy with cisplatin further enhances the survival outcome. Subsequent analysis reveals that POMC gene therapy inhibits the growth and metastasis of melanoma through apoptosis, angiogenesis inhibition, and modulation of epithelial-mesenchymal transition. Besides, £\-MSH/melanortin-1 receptor (MC-1R) pathway is involved in the POMC-mediated melanoma suppression. To investigate whether POMC therapy could be applied to other types of tumor, we evaluated the therapeutic efficacy of POMC gene therapy in Lewis lung carcinoma (LLC) cells which lack MC-1R. Interestingly, POMC gene delivery effectively inhibited the proliferation and colony formation of LLC cells in vitro and the growth of established LLC in mice. Histological analysis indicated that POMC gene delivery attenuated LLC through proliferation inhibition, apoptosis induction, and angiogenesis blockade. Moreover, POMC gene delivery perturbed £]-catenin signaling by reducing protein levels of £]-catenin and its downstream proto-oncogenes, including cyclin D1 and c-myc. These results support the existence of an MC-1R-independent pathway for POMC gene therapy and expand the therapeutic spectrum of POMC therapy for multiple types of cancer. To elucidate the role of host immunity in anti-neoplastic mechanism underlying POMC therapy, we compared the treatment efficacy of POMC gene therapy for B16-F10 melanoma between severe combined immune-deficient (SCID) and immune-competent C57BL/6 mice, and found similar extent of tumor suppression in both strains of mice. In addition, POMC gene therapy reduced the spleen weight and the number of circulating lymphocytes in B6 mice. These findings suggest that POMC therapy was not dependent on host immunity, yet instead induced immune suppression of animals through ACTH/cortisol production. To minimize such side effect of POMC therapy, we generated a series of adenovirus vectors encoding POMC with mutations in ACTH domain (ACTH-K15A/R17A), which fails to stimulate cortisol synthesis in vitro and in vivo. Gene delivery of ACTH (K15A/R17A) remained capable of suppressing the primary and metastatic melanoma, but had no effect on immune functions in mice. In conclusion, we have characterized the anti-neoplastic function and mechanism of POMC therapy for cancer. Furthermore, we have developed improved POMC gene vectors to minimize its adverse effect for future cancer therapy.

Despite intensive research on the neurobiological mechanisms of chronic pain, this therapeutic area remains one of the least satisfactorily covered by current drugs. Glutamate activates two major classes of receptors: ionotropic and metabotropic. Ionotropic receptors are classified into three major subclasses:a-amino-3- hydroxy-5-methyl-4- isoxazolepropionic acid (AMPA), kainate and N-methyl-D-aspartate (NMDA). NMDA receptor activation, at the level of the spinal cord and peripheral tissue has been shown to play an important role in the facilitation of nociption in several animal models. Although the efficacy of NMDA receptor antagonists in various experimental and clinical pain situations has been well documented, their use as analgesics is limited by serious side effects such as memory impairment, psychotomimetic effects, ataxia and motor incoordination. Two promising current approaches to obtain effective analgesia devoid of side effects are by subtype-selective NMDA receptor antagonism in central nervous system (CNS) or peripheral use of NMDA receptor antagonist that do not interfere with central glutamate processing. NR2B subunit of NMDA receptor was predominantly found in the superficial dorsal horn of spinal cord. Recent discoveries have revealed that the transfection of small interfering RNAs (siRNAs) into animal cells results in the potent, long-lasting post-transcriptional silencing of specific genes. Thus, two approaches of antagonizing NMDA receptor in CNS and peripheral nervous system (PNS) for pain relief using siRNAs or pharmacological agents are investigated in this study. The first approach involves intrathecal administration of NR2B-siRNA into subarachnoid space and transfection of siRNA into cell of spinal cord by transfection agent of polyethylenimine (PEI). Formalin test was used to induce inflammatory pain in the hind paw of rats. Behavior response to formalin test was observed and recorded on 3rd, 7th, 14th, and 21th day after injection of siRNA. The spinal cords were dissected immediately after formalin test and used for analysis of mRNA and protein. The results revealed that the use of siRNA targeting the NR2B subunit could abolish formalin induced pain behaviors and not impair motor coordination in rat model. The expression of NR2B mRNA and its associated protein as demonstrated by real time reverse transcription-polymerase chain reaction (RT-PCR) and western blotting were decreased. Significant reduction of NR2B immunoreactivity in dorsal horn of spinal cord were detected after 7 days treated by NR2B siRNA. The peak effect of gene knockdown occurred on day 3 for mRNA and day 7 for its protein, following intrathecal injection of 5 &#x00B5;g of siRNA targeting NR2B subunit. The inhibition of NR2B mRNA and protein lasted about 14 days and recovered on 21th days after injection of siRNA. The nociceptive response induced by formalin was decreased during the period of downregulation of NR2B protein. A novel intrathecal delivery of siRNA transfected with PEI into cell of dorsal horn reduced formalin-induced pain. The second approach involves subcutaneous injection of NMDA receptor antagonist and topical use of alpha2-adrenergic agonist for abolishing surgical pain. Additionally, we proved the upregulation of glutamate receptors in human inflamed skin. The study examined whether the peripheral ionotropic glutamate receptors (iGluRs) increased in inflamed human skin taken from patients having inflammatory pain over inflamed skin and surrounding area. Real time RT-PCR and western blot were used for quantitation of mRNA and protein of iGluR in normal and inflamed human skin. A significant increase in mRNA and protein for the subunits of NMDA, AMPA, and kainate receptor were detected in inflamed skin when compared to normal skin. The results demonstrate that mRNA and protein level of iGluRs are increasingly expressed during states of persistent inflammation, and that this increased activity may be involved in mediating clinical inflammatory pain in human skin. To examine the postoperative analgesic effect and adverse effect of local NMDA receptor antagonist (ketamine), ketamine (0.3%, 3 ml) or saline was subcutaneous infiltrated pre-incisionally in 26 patients equally assigned to two groups undergoing circumcision surgery. The saline-infiltrated subjects also received 9-mg intramuscular ketamine into the upper arm to control for any related systemic analgesic effects. The postoperative analgesic and adverse effects were followed for 24 hours. For ketamine infiltrated patients, the time interval until first analgesic demand was prolonged and the incidence of pain free (pain score = 0) during movement and erection was significant higher than saline infiltrated patients. No significant differences were noted in the incidence of adverse effects between the two groups. Pre-incisional subcutaneous infiltration of ketamine acting via a peripheral mechanism can suppression postoperative pain after circumcision surgery. Apraclonidine hydrochloride (AH) is a topical, relatively selective alpha2-adrenergic agonist that has limited access to the CNS and exhibits fewer systemic (adverse) effects such as dizziness and hypotension. Eighty patients scheduled for arthroscopic knee surgery received either intraarticular (IA) normal saline, 50 ug IA AH, 150 ug IA AH, or 150 ug IA clonidine subsequent to surgery. The IA application of 150 ug apraclonidine and 150 ug clonidine provide similar degree of postoperative analgesia and similar incidence of adverse effect. The promise is that both approaches attenuating nociception state devoid of CNS adverse effects provide novel approach for the management of chronic pain.