title: Induction of lgA secretion in the bovine mammary gland

authors: Hodgkinson, A.J.

abstract: This thesis investigates molecular and cellular mechanisms involved in lgA secretion in the bovine mammary gland. Our previous research showed that multi-site immunisation during late-pregnancy, involving priming by intra-peritoneal and intramuscular routes followed by intra-mammary immunisation, enhanced lgA secretion in milk and this effect was localised to immunised glands. This thesis addresses the hypothesis that increased lgA is produced by localised lgA plasma cells of mucosal origin that are recruited to the mammary gland by changes induced by intra-mammary immunisation. 
Recruitment mechanisms for lymphocytes were studied by examining expression of adhesion molecules MAdCAM-1, VCAM-1 and PNAd in mammary tissues of untreated animals, using immunohistochemical and molecular techniques. No immunoreactive MAdCAM-1 protein was found in venules and very low levels of MAdCAM-1 mRNA were found in tissues. By contrast, VCAM-1 protein was observed on larger venules. PNAd protein was only detected in supramammary lymph nodes, suggestive of a role for recruiting naïve lymphocytes. 
Cows were immunised with only one udder-half receiving intra-mammary immunisation to investigate effects on immunised glands. Animals with increased levels of lgA in milk had significantly more lgA plasma cells and T cells in immunised glands compared with non-immunised glands. Again no detectable MAdCAM-1 protein and only very low levels of MAdCAM-1 mRNA were found. No differences were observed in VCAM-1 protein expression between immunised and non-immunised mammary tissues. Together these studies suggest that MAdCAM-1 and VCAM-1 are not involved in lymphocyte recruitment to the bovine mammary gland, which contrasts to mice. 
In another study, cows categorised as either high- or low-responders to the multi-site immunisation were re-immunised to compare responses. Changes to lymphocytes in mammary secretions collected at intervals following two intra-mammary immunisations were monitored by flow cytometry. The percentage of T cells was found to decrease in both groups, although CD4 cells increased relative to CD8. The percentage of lgA-positive cells increased in a biphasic manner with high-responding animals showing an earlier and more sustained increase in lgA-positive cells compared with low-responders. The strong correlation observed between percentages of lgA-positive cells and lgA antibody in secretions suggests high levels of lgA require greater numbers of lgA-producing cells. 
Molecular analysis of known key cytokines found higher levels of IL-8 and IFN-𝛾 mRNA in immunised mammary tissues compared with non-immunised glands. In cells from mammary secretions, IL-8 mRNA increased shortly after immunisation while IFN- 𝛾 and TGF-β mRNA increased at a later stage. However, no discernible differences were observed between high- and low-responding animals. TNF-α, IL-2, IL-6 and IL-10 mRNA were found to be low in both mammary tissues and secretion cells. lgA mRNA was increased in immunised tissues although no correlation was observed between lgA mRNA and mRNA levels of the polyimmunoglobulin receptor. These analyses indicate that innate and cell-mediated responses were induced along with an antibody response to immunisation. 
Together these results support the hypothesis and indicate that intra-mammary immunisation increases lgA-producing cells in the mammary gland accompanied by changes in expression profiles of a range of cytokine genes. However, in contrast to mice, the data suggest little involvement of known adhesion molecules in recruiting lymphocytes to the gland.

text: Induction of lgA secretion in the bovine mammary gland This thesis investigates molecular and cellular mechanisms involved in lgA secretion in the bovine mammary gland. Our previous research showed that multi-site immunisation during late-pregnancy, involving priming by intra-peritoneal and intramuscular routes followed by intra-mammary immunisation, enhanced lgA secretion in milk and this effect was localised to immunised glands. This thesis addresses the hypothesis that increased lgA is produced by localised lgA plasma cells of mucosal origin that are recruited to the mammary gland by changes induced by intra-mammary immunisation. 
Recruitment mechanisms for lymphocytes were studied by examining expression of adhesion molecules MAdCAM-1, VCAM-1 and PNAd in mammary tissues of untreated animals, using immunohistochemical and molecular techniques. No immunoreactive MAdCAM-1 protein was found in venules and very low levels of MAdCAM-1 mRNA were found in tissues. By contrast, VCAM-1 protein was observed on larger venules. PNAd protein was only detected in supramammary lymph nodes, suggestive of a role for recruiting naïve lymphocytes. 
Cows were immunised with only one udder-half receiving intra-mammary immunisation to investigate effects on immunised glands. Animals with increased levels of lgA in milk had significantly more lgA plasma cells and T cells in immunised glands compared with non-immunised glands. Again no detectable MAdCAM-1 protein and only very low levels of MAdCAM-1 mRNA were found. No differences were observed in VCAM-1 protein expression between immunised and non-immunised mammary tissues. Together these studies suggest that MAdCAM-1 and VCAM-1 are not involved in lymphocyte recruitment to the bovine mammary gland, which contrasts to mice. 
In another study, cows categorised as either high- or low-responders to the multi-site immunisation were re-immunised to compare responses. Changes to lymphocytes in mammary secretions collected at intervals following two intra-mammary immunisations were monitored by flow cytometry. The percentage of T cells was found to decrease in both groups, although CD4 cells increased relative to CD8. The percentage of lgA-positive cells increased in a biphasic manner with high-responding animals showing an earlier and more sustained increase in lgA-positive cells compared with low-responders. The strong correlation observed between percentages of lgA-positive cells and lgA antibody in secretions suggests high levels of lgA require greater numbers of lgA-producing cells. 
Molecular analysis of known key cytokines found higher levels of IL-8 and IFN-𝛾 mRNA in immunised mammary tissues compared with non-immunised glands. In cells from mammary secretions, IL-8 mRNA increased shortly after immunisation while IFN- 𝛾 and TGF-β mRNA increased at a later stage. However, no discernible differences were observed between high- and low-responding animals. TNF-α, IL-2, IL-6 and IL-10 mRNA were found to be low in both mammary tissues and secretion cells. lgA mRNA was increased in immunised tissues although no correlation was observed between lgA mRNA and mRNA levels of the polyimmunoglobulin receptor. These analyses indicate that innate and cell-mediated responses were induced along with an antibody response to immunisation. 
Together these results support the hypothesis and indicate that intra-mammary immunisation increases lgA-producing cells in the mammary gland accompanied by changes in expression profiles of a range of cytokine genes. However, in contrast to mice, the data suggest little involvement of known adhesion molecules in recruiting lymphocytes to the gland.

year: 2005

title: Immobilization of [Pt₂(μ-S)₂(PPh₃)₄] on polymeric supports by sulfide alkylation and phosphine exchange reactions

authors: Ujam, Oguejiofo Theophilus

subjects: platinum complexes

abstract: The immobilization of the dinuclear platinum(II) sulfido complex [Pt₂(μ-S)₂ (PPh₃)₄] on solid supports has been investigated. Reaction with haloalkyl functionalized polymers [Merrifield's resin (chloromethylated polystyrene), chloropropyl silica, chloropropyl controlled pore glass, and bromopropyl polysiloxane] gives complexes immobilized through alkylation of one of the sulfide ligands, forming a μ-thiolate ligand acting as an anchor to the polymer support, akin to well-established reactions of [Pt₂(μ-S)₂(PPh₃)₄] with molecular alkylating agents. The model complex [Pt₂(μ-S)(μ-SCH₂SiMe₃)(PPh₃)₄]PF₆ was prepared as the first molecular silicon-containing derivative of [Pt₂(μ-S)₂(PPh₃)₄] and was fully characterized by NMR spectroscopy, electrospray ionization-mass spectrometry, and single-crystal X-ray diffraction. Immobilization of [Pt₂(μ-S)₂(PPh₃)₄] by phosphine exchange reactions was also achieved using commercial polystyrene-grafted triphenylphosphine or a new immobilized phosphine [derived by sequential functionalization of Merrifield's resin with a polyether amine and then Ph₂PCH₂OH].

text: Immobilization of [Pt₂(μ-S)₂(PPh₃)₄] on polymeric supports by sulfide alkylation and phosphine exchange reactions The immobilization of the dinuclear platinum(II) sulfido complex [Pt₂(μ-S)₂ (PPh₃)₄] on solid supports has been investigated. Reaction with haloalkyl functionalized polymers [Merrifield's resin (chloromethylated polystyrene), chloropropyl silica, chloropropyl controlled pore glass, and bromopropyl polysiloxane] gives complexes immobilized through alkylation of one of the sulfide ligands, forming a μ-thiolate ligand acting as an anchor to the polymer support, akin to well-established reactions of [Pt₂(μ-S)₂(PPh₃)₄] with molecular alkylating agents. The model complex [Pt₂(μ-S)(μ-SCH₂SiMe₃)(PPh₃)₄]PF₆ was prepared as the first molecular silicon-containing derivative of [Pt₂(μ-S)₂(PPh₃)₄] and was fully characterized by NMR spectroscopy, electrospray ionization-mass spectrometry, and single-crystal X-ray diffraction. Immobilization of [Pt₂(μ-S)₂(PPh₃)₄] by phosphine exchange reactions was also achieved using commercial polystyrene-grafted triphenylphosphine or a new immobilized phosphine [derived by sequential functionalization of Merrifield's resin with a polyether amine and then Ph₂PCH₂OH].

year: 2013

title: He Tangata Kei Tua: Guidelines for biobanking with Māori.

authors: Hudson, Maui

abstract: Māori ethical frameworks recognise that all research in New Zealand is of interest to Māori and outline community expectations of appropriate behavior in research to deliver the best outcomes for Māori. Research contributes to the broader development objectives of society and this endeavor is being supported by biobanking infrastructure. Ethics has a specific role in guiding key behaviours, processes and methodologies used in research.
This document outlines a framework for addressing Māori ethical issues within the context of biobanking. It draws on a foundation of mātauranga (Indigenous knowledge) and tikanga Māori (Māori protocols and practices) and will be useful for researchers, ethics committee members and those who engage in consultation or advice about biobanking with Māori in local, regional, national or international settings.

text: He Tangata Kei Tua: Guidelines for biobanking with Māori. Māori ethical frameworks recognise that all research in New Zealand is of interest to Māori and outline community expectations of appropriate behavior in research to deliver the best outcomes for Māori. Research contributes to the broader development objectives of society and this endeavor is being supported by biobanking infrastructure. Ethics has a specific role in guiding key behaviours, processes and methodologies used in research.
This document outlines a framework for addressing Māori ethical issues within the context of biobanking. It draws on a foundation of mātauranga (Indigenous knowledge) and tikanga Māori (Māori protocols and practices) and will be useful for researchers, ethics committee members and those who engage in consultation or advice about biobanking with Māori in local, regional, national or international settings.

year: 2016