The identification of a novel human integrin $/beta$ subunit, $/beta/sb7$, has been reported in this dissertation. The full-length human $/beta/sb7$ cDNA contains 2798 nucleotides encoding a single long open reading frame of 798 amino acid residues of which 708 are extracellular and 52 are cytoplasmic. The human $/beta/sb7$ sequence displays strong homology with other integrin $/beta$ subunits, being most related to $/beta/sb2$. Cell distribution studies suggest that the expression of transcripts encoding the human $/beta/sb7$ subunit is restricted to leukocytes. This study was the first to show that the cysteine-rich repeats of the human integrin $/beta/sb7$ subunit and other integrin $/beta$ subunits are related to domain III of the laminin B chains. Furthermore, the integrin $/beta$ subunit and laminin cysteine-rich repeats are each related to the epidermal growth factor (EGF)-like repeats found in a growing number of adhesion molecules and regulatory proteins. The mouse homologue of the human $/beta/sb7$ subunit has also been cloned in this study, and it shares 87% amino acid identity with the human $/beta/sb7$ sequence. The mouse $/beta/sb7$ mRNA transcripts, like that of the human $/beta/sb7$, have a predominantly leukocyte-restricted expression, but in some haematopoietic lineage cell lines, in addition to the predominant 3 kb transcript, a less abundant 2 kb transcript is also detectable. Cultured mouse skin epithelial cells contain only the 2 kb transcript. The genes encoding the human and mouse $/beta/sb7$ subunits were mapped to human chromosome 12 (ITGB7) and mouse chromosome 15 (Itgb-7). The Itgb-7 gene was localised to the distal part of mouse chromosome 15, and exists in the gene order: Myc-Wnt-1-Itgb-1. The N-terminal 13 amino acids of the mature mouse $/beta/sb7$ subunit was shown to be identical to a 13 amino acid N-terminal sequence reported for the Mr 120,000 $/beta$ subunit of the M290 antigen, which is found highly expressed on mouse intestinal intraepithelial lymphocytes (IEL). This finding suggests that the integrin $/beta/sb7$ subunit may play important roles in IEL migration, and immunosurveillance in the gut mucosa. / Subscription resource available via Digital Dissertations only.

The identification of a novel human integrin $/beta$ subunit, $/beta/sb7$, has been reported in this dissertation. The full-length human $/beta/sb7$ cDNA contains 2798 nucleotides encoding a single long open reading frame of 798 amino acid residues of which 708 are extracellular and 52 are cytoplasmic. The human $/beta/sb7$ sequence displays strong homology with other integrin $/beta$ subunits, being most related to $/beta/sb2$. Cell distribution studies suggest that the expression of transcripts encoding the human $/beta/sb7$ subunit is restricted to leukocytes. This study was the first to show that the cysteine-rich repeats of the human integrin $/beta/sb7$ subunit and other integrin $/beta$ subunits are related to domain III of the laminin B chains. Furthermore, the integrin $/beta$ subunit and laminin cysteine-rich repeats are each related to the epidermal growth factor (EGF)-like repeats found in a growing number of adhesion molecules and regulatory proteins. The mouse homologue of the human $/beta/sb7$ subunit has also been cloned in this study, and it shares 87% amino acid identity with the human $/beta/sb7$ sequence. The mouse $/beta/sb7$ mRNA transcripts, like that of the human $/beta/sb7$, have a predominantly leukocyte-restricted expression, but in some haematopoietic lineage cell lines, in addition to the predominant 3 kb transcript, a less abundant 2 kb transcript is also detectable. Cultured mouse skin epithelial cells contain only the 2 kb transcript. The genes encoding the human and mouse $/beta/sb7$ subunits were mapped to human chromosome 12 (ITGB7) and mouse chromosome 15 (Itgb-7). The Itgb-7 gene was localised to the distal part of mouse chromosome 15, and exists in the gene order: Myc-Wnt-1-Itgb-1. The N-terminal 13 amino acids of the mature mouse $/beta/sb7$ subunit was shown to be identical to a 13 amino acid N-terminal sequence reported for the Mr 120,000 $/beta$ subunit of the M290 antigen, which is found highly expressed on mouse intestinal intraepithelial lymphocytes (IEL). This finding suggests that the integrin $/beta/sb7$ subunit may play important roles in IEL migration, and immunosurveillance in the gut mucosa. / Subscription resource available via Digital Dissertations only.

The identification of a novel human integrin $/beta$ subunit, $/beta/sb7$, has been reported in this dissertation. The full-length human $/beta/sb7$ cDNA contains 2798 nucleotides encoding a single long open reading frame of 798 amino acid residues of which 708 are extracellular and 52 are cytoplasmic. The human $/beta/sb7$ sequence displays strong homology with other integrin $/beta$ subunits, being most related to $/beta/sb2$. Cell distribution studies suggest that the expression of transcripts encoding the human $/beta/sb7$ subunit is restricted to leukocytes. This study was the first to show that the cysteine-rich repeats of the human integrin $/beta/sb7$ subunit and other integrin $/beta$ subunits are related to domain III of the laminin B chains. Furthermore, the integrin $/beta$ subunit and laminin cysteine-rich repeats are each related to the epidermal growth factor (EGF)-like repeats found in a growing number of adhesion molecules and regulatory proteins. The mouse homologue of the human $/beta/sb7$ subunit has also been cloned in this study, and it shares 87% amino acid identity with the human $/beta/sb7$ sequence. The mouse $/beta/sb7$ mRNA transcripts, like that of the human $/beta/sb7$, have a predominantly leukocyte-restricted expression, but in some haematopoietic lineage cell lines, in addition to the predominant 3 kb transcript, a less abundant 2 kb transcript is also detectable. Cultured mouse skin epithelial cells contain only the 2 kb transcript. The genes encoding the human and mouse $/beta/sb7$ subunits were mapped to human chromosome 12 (ITGB7) and mouse chromosome 15 (Itgb-7). The Itgb-7 gene was localised to the distal part of mouse chromosome 15, and exists in the gene order: Myc-Wnt-1-Itgb-1. The N-terminal 13 amino acids of the mature mouse $/beta/sb7$ subunit was shown to be identical to a 13 amino acid N-terminal sequence reported for the Mr 120,000 $/beta$ subunit of the M290 antigen, which is found highly expressed on mouse intestinal intraepithelial lymphocytes (IEL). This finding suggests that the integrin $/beta/sb7$ subunit may play important roles in IEL migration, and immunosurveillance in the gut mucosa. / Subscription resource available via Digital Dissertations only.

The identification of a novel human integrin $/beta$ subunit, $/beta/sb7$, has been reported in this dissertation. The full-length human $/beta/sb7$ cDNA contains 2798 nucleotides encoding a single long open reading frame of 798 amino acid residues of which 708 are extracellular and 52 are cytoplasmic. The human $/beta/sb7$ sequence displays strong homology with other integrin $/beta$ subunits, being most related to $/beta/sb2$. Cell distribution studies suggest that the expression of transcripts encoding the human $/beta/sb7$ subunit is restricted to leukocytes. This study was the first to show that the cysteine-rich repeats of the human integrin $/beta/sb7$ subunit and other integrin $/beta$ subunits are related to domain III of the laminin B chains. Furthermore, the integrin $/beta$ subunit and laminin cysteine-rich repeats are each related to the epidermal growth factor (EGF)-like repeats found in a growing number of adhesion molecules and regulatory proteins. The mouse homologue of the human $/beta/sb7$ subunit has also been cloned in this study, and it shares 87% amino acid identity with the human $/beta/sb7$ sequence. The mouse $/beta/sb7$ mRNA transcripts, like that of the human $/beta/sb7$, have a predominantly leukocyte-restricted expression, but in some haematopoietic lineage cell lines, in addition to the predominant 3 kb transcript, a less abundant 2 kb transcript is also detectable. Cultured mouse skin epithelial cells contain only the 2 kb transcript. The genes encoding the human and mouse $/beta/sb7$ subunits were mapped to human chromosome 12 (ITGB7) and mouse chromosome 15 (Itgb-7). The Itgb-7 gene was localised to the distal part of mouse chromosome 15, and exists in the gene order: Myc-Wnt-1-Itgb-1. The N-terminal 13 amino acids of the mature mouse $/beta/sb7$ subunit was shown to be identical to a 13 amino acid N-terminal sequence reported for the Mr 120,000 $/beta$ subunit of the M290 antigen, which is found highly expressed on mouse intestinal intraepithelial lymphocytes (IEL). This finding suggests that the integrin $/beta/sb7$ subunit may play important roles in IEL migration, and immunosurveillance in the gut mucosa. / Subscription resource available via Digital Dissertations only.

The identification of a novel human integrin $/beta$ subunit, $/beta/sb7$, has been reported in this dissertation. The full-length human $/beta/sb7$ cDNA contains 2798 nucleotides encoding a single long open reading frame of 798 amino acid residues of which 708 are extracellular and 52 are cytoplasmic. The human $/beta/sb7$ sequence displays strong homology with other integrin $/beta$ subunits, being most related to $/beta/sb2$. Cell distribution studies suggest that the expression of transcripts encoding the human $/beta/sb7$ subunit is restricted to leukocytes. This study was the first to show that the cysteine-rich repeats of the human integrin $/beta/sb7$ subunit and other integrin $/beta$ subunits are related to domain III of the laminin B chains. Furthermore, the integrin $/beta$ subunit and laminin cysteine-rich repeats are each related to the epidermal growth factor (EGF)-like repeats found in a growing number of adhesion molecules and regulatory proteins. The mouse homologue of the human $/beta/sb7$ subunit has also been cloned in this study, and it shares 87% amino acid identity with the human $/beta/sb7$ sequence. The mouse $/beta/sb7$ mRNA transcripts, like that of the human $/beta/sb7$, have a predominantly leukocyte-restricted expression, but in some haematopoietic lineage cell lines, in addition to the predominant 3 kb transcript, a less abundant 2 kb transcript is also detectable. Cultured mouse skin epithelial cells contain only the 2 kb transcript. The genes encoding the human and mouse $/beta/sb7$ subunits were mapped to human chromosome 12 (ITGB7) and mouse chromosome 15 (Itgb-7). The Itgb-7 gene was localised to the distal part of mouse chromosome 15, and exists in the gene order: Myc-Wnt-1-Itgb-1. The N-terminal 13 amino acids of the mature mouse $/beta/sb7$ subunit was shown to be identical to a 13 amino acid N-terminal sequence reported for the Mr 120,000 $/beta$ subunit of the M290 antigen, which is found highly expressed on mouse intestinal intraepithelial lymphocytes (IEL). This finding suggests that the integrin $/beta/sb7$ subunit may play important roles in IEL migration, and immunosurveillance in the gut mucosa. / Subscription resource available via Digital Dissertations only.

Distant metastases are a limiting factor in cancer patient survival as they are least accessible to conventional therapies. Effective therapy should treat primary tumours and metastatic disease. Use of image-guided radiation therapy (IGRx) enables high doses of radiation to be delivered for better tumour control while minimizing toxicity to healthy tissues. Systemic effects on distant non-irradiated tissues have been observed following IGRx. This phenomenon, termed the abscopal effect, is hypothesized to be mediated by the immune system. The inflammatory milieu generated following IGRx may activate immune cells to mount specific anti-tumour responses. The work described in this thesis aims to develop a model to study the abscopal effect, and evaluate the potential of combining IGRx and immunotherapy to enhance such distant tumour killing. Results from these studies may have clinical implications, where a combined IGRx and immunotherapy approach may prove useful in eliciting regression of local tumours and distant metastases.

Distant metastases are a limiting factor in cancer patient survival as they are least accessible to conventional therapies. Effective therapy should treat primary tumours and metastatic disease. Use of image-guided radiation therapy (IGRx) enables high doses of radiation to be delivered for better tumour control while minimizing toxicity to healthy tissues. Systemic effects on distant non-irradiated tissues have been observed following IGRx. This phenomenon, termed the abscopal effect, is hypothesized to be mediated by the immune system. The inflammatory milieu generated following IGRx may activate immune cells to mount specific anti-tumour responses. The work described in this thesis aims to develop a model to study the abscopal effect, and evaluate the potential of combining IGRx and immunotherapy to enhance such distant tumour killing. Results from these studies may have clinical implications, where a combined IGRx and immunotherapy approach may prove useful in eliciting regression of local tumours and distant metastases.

Doctor of Philosophy / Department of Anatomy and Physiology / Elizabeth G. Davis / Activation of innate immunity through Toll-like receptor (TLR) signaling can also enhance antigen-specific adaptive immunity. TLR9 is an endosomal receptor for unmethylated bacterial and viral cytosine-phosphate-guanine DNA (CpG-DNA). West Nile virus (WNV) infection may result in meningitis and encephalitis in humans and horses, especially aged and immunocompromised individuals. Using flow cytometric analyses and quantitative reverse transcriptase-polymerase chain reaction (RT-PCR), we investigated equine cell-mediated immunity (CMI) to an inactivated West Nile virus vaccine in healthy yearling and adult horses. We also studied the potential of enhancing equine adaptive immunity to viruses and other pathogens by activation of innate immunity though TLR9 signaling pathway. We found vaccination with inactivated WNV vaccine induced strong WNV-specific T helper type 1 (Th1) and Th2 CMI with a Th1 bias, also effectively induced WNV-specific CTLs in yearling horses. In adult horses, the pre-existing Th1 CMI bias against WNV was enhanced following booster vaccination with inactivated WNV vaccine. Molecular characterization and flow cytometric analysis of TLR9 expression using a cross-reactive TLR9 mAb identified high constitutive expression of equine TLR9 in neutrophils (PMNs), CD4[superscript]+ and CD8[superscript]+ T cells and other leukocytes. Conservation of equine TLR9 and a high expression profile among leukocytes suggests that equine TLR9 is a frequent target for unmethylated CpG-DNA, an essential mechanism for the activation of innate immunity. Unmethylated CpG-DNA can significantly activate equine PMNs. It also induces expression of interferon (IFN)-[Alpha], IFN-[Beta], IFN-[Gamma], and interleukin (IL)-12p35 in PBMCs, as well as IFN-[alpha] and IFN-[gamma] in monocyte-derived DCs. Enhanced expression of IFNs in immune cells by CpG-DNA is not only crucial for host viral clearance, but also important in mediating host immune responses due to IFNs' anti-inflammatory effects. Compared to the relatively weaker activation of equine innate immunity by inactivated WNV, the tested CpG-DNA species showed potential as vaccine adjuvants for enhancement of CTLs and Th1 CMI against intracellular pathogens, characterized by significant induction of type I IFNs and Th1-specific cytokines such as IL-12p35 and IFN-γ. These data provide a basis for further investigation of these CpG-DNA species as potentially effective vaccine adjuvants in horses.

Master of Science / Department of Diagnostic Medicine and Pathobiology / Juergen A. Richt / Influenza A virus is an important respiratory pathogen with the potential to affect both humans and animals, thereby creating the conditions for public health disasters, especially during pandemic episodes. At present, two primary strategies to combat influenza are vaccination and antiviral drugs. Since influenza viruses mutate rapidly and constantly via antigenic drift and shift, vaccines can become quickly outdated; and resistance to antiviral drugs can readily result. Interferon alpha (IFN-[alpha]) plays an important role as a first line of innate antiviral immunity. To investigate the antiviral potential of exogenously applied IFN-[alpha] on the replication of different subtypes of influenza A viruses, three subtypes of influenza A virus, i.e. swine H3N2, pandemic H1N1 and avian H9N2 were chosen. Their replication kinetics in the presence of Alferon N (human Interferon alpha) on human epithelium (A549) cells and swine testis (ST) cells was evaluated. In these tests of the three subtypes of influenza A viruses, it was found that the replication ability of all three viruses was inhibited when ST cells were treated with Alferon for four hours before infection. The ability of Alferon to inhibit influenza A viruses replication was found to be dose-dependent. Similar results were obtained when A549 cells were used; however, pretreatment of A549 cells with Alferon for more than 16 hours was necessary before infection. Furthermore, the expression of some ISGs (Interferon stimulated genes) between ST and A549 cells was also investigated. The differences in response of the ISGs between the two cell lines provided an explanation of the disparity towards exogenous interferon treatment. In summary, these results demonstrated that Alferon N has the ability to inhibit replication of different subtypes of influenza A viruses in cell cultures. This study provides a foundation for future in vivo studies using exogenous IFN-[alpha] treatment as an alternative approach to combat influenza A virus infection.

Master of Science / Division of Biology / Sherry D. Fleming / Professional phagocytes play a critical role in maintaining homeostasis within a host through phagocytic, microbicidal, and inflammatory activity. Complement receptors (CR) and toll-like receptors (TLRs) aid in phagocytosis and stimulate these cells to enhance the immune response. Environmental factors such as hypoxia, prevalent at sites of tissue damage or infection, induce a similar effect. Systemic components such as opsonins may further enhance phagocyte activity. Similar to professional phagocytes, B1 B cells exhibit a broad range of immunological activity as well as expression of CRs and TLRs. Despite extensive studies with other phagocytes, the effects of CRs and TLRs expression, hypoxic stimulation, or opsonization on B1 B cell function remain unclear. We tested the hypothesis that TLR2 stimulation, hypoxia, CR2 expression, or opsonins would enhance B1 B cell phagocytic and inflammatory activity. Negatively selected peritoneal cavity B1 B cells from the (PerC) of wild type, Tlr2[superscript]-[superscript]/[superscript]-, and Cr2[superscript]-[superscript]/[superscript]- mice, or a B1 B-like cell line, Wehi 231, were subjected to normoxia or hypoxia with or without particles for phagocytosis, TLR2 agonists, or CR2 ligands. The PerC of Tlr2[superscript]-[superscript]/[superscript]- mice contained an altered B1 B cell subset distribution while Cr2[superscript]-[superscript]/[superscript]- mice exhibited a normal repertoire. We demonstrated that hypoxia significantly downregulated inflammatory cytokine production by B1 B cells, while upregulating phagocytic activity in a TLR2 or CR2 dependent manner. TLR2 or CR2 deficiency altered constitutive production of B1 B cell associated cytokines. The CR2 ligand C3d, an opsonin, significantly enhanced the phagocytic activity of B1 B cells but failed to stimulate cytokine production. However, Cr2[superscript]-[superscript]/[superscript]- B1 B cells phagocytosed C3d-coated particles suggesting multiple CR may play a role in B1 B cell phagocytosis. Overall, the data suggest TLRs, CRs, hypoxia, and opsonization all contribute to B1 B cell effector function similar to professional phagocytes.