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.

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.

Doctor of Philosophy / Department of Anatomy and Physiology / Michael J. Kenney / Important bidirectional interactions exist between the central nervous system and the immune system. Neural-immune interactions provide a regulatory system in the body and disturbances in these interactions may lead to disease. Although the sympathetic nervous system is thought to play a key role in mediating neural-immune interactions, central neural mechanisms mediating sympathetic-immune interactions and the effect of centrally-induced alterations in sympathetic nerve discharge on immune function is not known. We tested the hypothesis that central activation of sympathetic neural circuits alters splenic cytokine gene expression. In a separate study, we tested the hypothesis that hypothermia-induced changes in visceral sympathetic nerve discharge (SND) would be attenuated in middle-aged and aged compared with young rats. Previous studies have demonstrated that skin sympathoexcitatory responses to skin cooling are attenuated in aged compared with young subjects, suggesting that advancing age influences sympathetic nerve responsiveness to cooling. The effect of age on sympathetic nerves innervating other targets organs during acute cooling remains unknown. Central activation of splenic SND was produced using three different experimental interventions: increased core body temperature produced by acute heating, intracerebroventricular injection of angiotensin II (ANGII), and decreased core body temperature produced by acute cooling. Changes in gene expression profiles were analyzed using inflammatory cytokine-specific gene-array and further validated using real-time RT-PCR analysis. The following observations were made. 1)Splenic SNDincreased in response to each experimental intervention except in acute cooled young rats where there was a decrease in splenic SND. 2) Splenic cytokine gene expression of pro-inflammatory cytokines (e.g., IL-1β, IL-6, IL-2) and chemokines (GRO1, CXCL2, CCCL2 and, CXCL10) was increased in response to each experimental intervention. 3) Expression of splenic cytokine genes was reduced after splenic-denervation except in acute cooled rats. 4) Progressive hypothermia reduced splenic, renal, and adrenal SND in rats and was generally attenuated in middle-aged and aged rats. These results demonstrate the functional significance of changes in sympathetic nerve activity on splenic immune cell activation and the effect of age on SND responses to core body cooling.

Doctor of Philosophy / Department of Anatomy and Physiology / Chris R. Ross / The immediate antiviral defense residing in the innate immune system of multicellular organisms critically determines the outcome of viral infection. This dissertation presents a study of the "effectors" and "receptors" of porcine innate immunity in infection caused by porcine reproductive and respiratory syndrome virus (PRRSV), which is the most devastating pathogen impacting the swine industry. In the first investigation, eleven novel porcine host defense peptides (HDPs), [Beta]-defensins (pBDs), were identified and characterized. All of these peptides have a consensus [Beta]-defensin motif and phylogenetically are similar to orthologs from other species. A differential expression pattern for these 11 newly identified genes was found. For example, pBD-2 and pBD-3 were expressed in bone marrow, lung, skin and other lymphoid tissues. pBD-2 and pBD-3 were further characterized for their gene structure, and antimicrobial activity of synthetic peptides. The second study was conducted to evaluate PRRSV-induced differential expression of porcine HDPs and direct antiviral activity of selected HDPs against PRRSV. In vitro incubation of PRRSV with synthetic pBD-3 or protegrin-4 (PG-4) significantly inhibited viral infectivity. Using nine protegrin-derived peptides, it was determined that cyclization of PG-4 increased anti-PRRSV activity and mutation of some residues in PG-4 diminished some of the activity. These findings suggest the potential role of porcine HDPs as a group of innate antiviral effectors. In the third and fourth investigations, porcine Toll-like receptor (TLR) 3 and TLR7 were identified and functionally expressed. Increased expression of TLR3 was observed in PRRSV-infected porcine lungs. Stimulation of porcine alovelar macrophages with poly (I:C), a synthetic TLR3 ligand, increased expression of interferon-[Beta] and suppressed PRRSV infectivity. Activation of porcine TLR3 overexpressed in a PRRSV-sensitive cell line, elicited antiviral responses to PRRSV infection. Partial silencing of TLR3 in PAMs resulted in increased PRRSV infection. In summary, these data provide molecular information on porcine TLR3 and TLR7, and their involvement in PRRSV pathogenesis, which may elicit new strategies to prevent this costly swine disease.