Thesis (M. Phil.)--University of Hong Kong, 2000. / Includes bibliographical references (leaves 62-76).

Thesis (M.Phil.)--University of Hong Kong, 2000. / Includes bibliographical references (leaves 62-76) Also available in print.

published_or_final_version / Zoology / Master / Master of Philosophy

No description available.

With asphyxia, newborns may suffer cardiogenic shock with myocardial dysfunction and dysregulation of vasomotor tone resulting in multiorgan dysfunction. Vasoactive medications are often administered with limited evidence directing clinicians regarding the use of high-dose monotherapy with dopamine relative to combination treatment with dopamine and a second different agent. We hypothesized that the treatment of hypoxia-reoxygenated newborn piglets with combinations of vasoactive medications would improve systemic and regional hemodynamics. Instrumented newborn piglets were subjected to hypoxia-reoxygenation with subsequent infusion of high-dose dopamine or moderate-dose dopamine and one of epinephrine, milrinone or levosimendan. Treatment with high-dose dopamine improved systemic and mesenteric perfusion. The addition of low-dose epinephrine showed some benefits regarding pulmonary hypertension and should a non-catecholamine agent be added to dopamine, milrinone is preferred to levosimendan given benefits to mesenteric perfusion. We conclude that the selection of appropriate vasoactive medical therapy should be directed by the clinical effects desired. / Experimental Surgery

Diss. (sammanfattning) Stockholm : Karol. inst., 2004. / Härtill 5 uppsatser.

Thesis (MScMedSc)--Stellenbosch University, 2015. / ENGLISH ABSTRACT: Introduction: Chronic HIV-1 infection is characterized by immune activation and dysregulation of immune homeostasis, which impacts on multiple immune cell types. The B-cell compartment, which plays an important role in the producing neutralizing antibodies, is also dysregulated in HIV- 1 infection. In this study we investigated peripheral blood B-cell subset distribution, and changes in expression of cellular activation, inhibition, and apoptosis signaling markers in both untreated chronic HIV-1 infected individuals and healthy uninfected controls. The neuropeptide immune modulator, vasoactive intestinal peptide (VIP) is known to selectively down-regulate activation of CD4+ T-cells in various disease settings including HIV-1, however to our knowledge, no studies have investigated the effect of VIP inhibition on B-cell activation. Materials & Methods: A total of 21 HIV+ve (CD4 count >250 cells/µl), and 19 HIV-ve individuals were recruited from the Emavundleni voluntary testing and counseling clinic in Crossroads, Western Province, South Africa. Whole blood was stained to distinguish B-cell subsets (activated memory (AM: CD21-CD27+), resting memory (RM: CD21+CD27+), mature naïve (MN: CD21+CD27-), or tissue-like memory (TLM: CD21loCD27lo). In addition expression of markers of B-cell activation (CD126, CD86, CD38, CD284, CD287), inhibition (CD72, CD85j, CD300a, CD305, CD307d), and apoptosis signaling (CD95), was assessed ex vivo by flow cytometry (BD FACSCanto II). For determination of functional responsiveness isolated B-cells (RosetteSep, Stemcell Technologies) were cultured for 18h (37°C, 5%CO2) without stimulation or stimulated with TLR ligands (LPS or R848). Stimulation experiments were also performed in the presence or absence of VIP. Results: Chronic HIV-1 infection affected B-cell subset distribution. The percentage (%) of TLM was increased by 59.24%, and %RM was decreased by 22.73% (both p<0.01). Total expression of the VIP receptor VPAC2 was decreased by 47.35% (p=0.0296). Subsets had a mixed phenotype ex vivo; HIV infection upregulated CD38 (by 59.56%, p=0.0004), CD72 (by 60.70%, p=0.0396), CD307d (by 68.63%, p=0.0015) on AM, while RM B-cells had increased expression of TLR4 (by 107.04%, p=0.0057) and TLR7 (by 208.14%, p=0.0199). TLM B cells (i.e. exhausted phenotype) displayed upregulated TLR7 (by 550%, p=0.0128) and CD307d (by 72.40% p=0.045) expression. MN B-cells had increased CD72 expression (by 70.98%, p=0.0026). R848 upregulated CD86 expression by 42.20% on AM (p<0.01), and by 56.06% on RM B-cells (p<0.01), which was significantly downregulated with VIP inhibition (both p<0.05). Similarly, CD95 expression on RM, TLM, and MN B-cells increased by 31.10% (p<0.001), 21.46% (p<0.01), and 39.92% (p<0.01) with R848 stimulation respectively, which was also significantly downregulated with VIP inhibition. Conclusion: These data indicate that B-cells in untreated HIV infection display increased levels of activation, and also the potential for increased susceptibility to apoptosis as evidenced by increased FAS (CD95) expression. VIP significantly down-regulated markers of activation, inhibition, and apoptosis signaling. Dysregulation of B-cells is thus apparent in asymptomatic stable chronic HIV-1 infection, which may impact on both inefficient neutralizing antibody production and hypergammaglobulinemia. The ability of VIP to prevent stimulationassociated marker upregulation may indicate that VIP is a potential therapeutic agent. Its immuno-modulatory properties were demonstrated to limit B-cell hyperactivation, and selectively down-regulate apoptosis and mark it out for further investigation. / AFRIKAANSE OPSOMMING: Inleiding: Immunaktivering en ongekoppelde immuun-homeostase is kenmerke van chroniese MIVinfeksie. Ons het perifere bloed B-sel subgroep-verspreiding, en veranderinge in die uitdrukking van merkers van aktivering, inhibisie, en apoptose in 'n onbehandelde MIV-1 besmettende groep ondersoek (in vergelyk met 'n gesonde onbesmettende kontrole). Die immuun-moduleerder, vasoaktiewe intestinale peptied (VIP) is bekend om aktivasie van geaktiveerde CD4+ T-selle te verminder, maar tot ons kennis, is daar geen studies wat die effek van VIP-inhibisie op B-sel aktivering ondersoek het, in die konteks van MIV-1 infeksie. Materiaal & Metodes: MIV+we individue (CD4-telling >250 selle/µl) , en MIVwe kontroles is gewerf uit die vrywillige toetsing en berading Emavundleni kliniek, Crossroads, Westelike Provinsie, Suid-Afrika. Bsel subgroepe is gedefinieer as geaktiveerde geheue (AM: CD21- CD27+ ), rusende geheue (RM: CD21+ CD27+ ), volwasse naïef (MN: CD21+ CD27- ), of weefsel-agtige geheue (TLM: CD21loCD27lo). Merkers van aktivering (CD126, CD86, CD38, CD284, CD287), inhibisie (CD72, CD85j, CD300a, CD305, CD307d), en apoptose signalering (CD95) is via vloeisitometrie (BD FACSCanto II) op B-selle ex vivo en ook op geïsoleerde B-selle (RosetteSep, Cell Technologies) ondersoek. Vir die bepaling van funksionele responsiwiteit, geïsoleerde B-selle (RosetteSep, StemCell Technologies) was vir 18h (37°C, 5%CO2) gekweek, sonder stimulasie of gestimuleer met TLR ligande (LPS of R848). Stimulasie eksperimente het ook in die teenwoordigheid of afwesigheid van VIP plaasgevind. Resultate: Chroniese MIV-1 infeksie het B-sel subset verspreiding geraak. Die persentasie (%) van TLM is verhoog deur 59,24%, en% RM het met 22.73% afgeneem (beide p <0,01). Totale uitdrukking van die VIP reseptor VPAC2 het met 47,35% afgeneem (p = 0,0296). Subgroepe het 'n gemengde ex vivo fenotipe; MIV-infeksie het CD38 (deur 59,56%, p=0,0004), CD72 (deur 60,70%, p=0,0396), CD307d (deur 68,63%, p=0,0015) op AM verhoog, terwyl RM Bselle het verhoogde uitdrukking van TLR4 (deur 107,04%, p=0,0057) en TLR7 (deur 208,14%, p=0,0199). TLM B-selle (die uitgeputtende fenotiep) het verhoogde TLR7 (deur 550%, p=0,0128) en CD307d (deur 72,40% p=0.045) uitdrukking gewys. MN B-selle het verhoogde uitdrukking van CD72 (deur 70,98%, p = 0,0026). R848 het CD86 uitdrukking op AM deur 42,20%, en op RM deur 56,06% toegeneem (beide p <0,01). Dit het met VIP inhibisie beduidend afgeneem (beide p <0.05). CD95 uitdrukking was soortgelyk verhoog op RM, TLM, en MN B-selle met 31.10% (p <0.001), 21,46% (p <0,01), en 39,92% (p <0,01) met R848 stimulasie. Al drie het beduidend afgeneem met VIP inhibisie. Gevolgtrekking: Hierdie data dui daarop dat B-selle in onbehandelde MIV-infeksie vertoon verhoogde aktiveringsvlakke, en ook die potensiaal vir verhoogde vatbaarheid vir apoptose soos bewys deur verhoogde uitdrukking van FAS (CD95). VIP het beduidend merkers van aktivering, inhibisie, en apoptose af-gereguleer. Wanfunksie van B-selle is dus in asimptomatiese stabiele chroniese MIV-1 infeksie duidelik, wat impak kan hê op beide oneffektiewe neutraliserende teenliggaampie produksie, en hiepergammaglobulinimie. Die vermoë van VIP stimulasie-verwante merker opregulasie te voorkom kan aandui dat VIP 'n potensiële terapeutiese agent is. VIP se immuno-moduleerende eiendomme is gedemonstreer om Bsel hieperaktiveering te beperk, en selektief apoptose afreguleer, en merk dit vir verdere ondersoek.

Tryptases are trypsin-like enzymes found in mast cell granules. Although in vivo substrates have not been positively identified, tryptases cleave a limited number of potential physiological substrates in vitro, including high molecular weight kininogen (HMWK) and vasoactive intestinal peptide (VIP). Purified human lung mast cell tryptase (HLT) apparently exists as a tetramer with an M$\sb{\rm r}$ of 135-144 kDa by gel filtration, whereas SDS-PAGE yielded two bands of M$\sb{\rm r}$ 29 Kda and 33 Kda. Tryptases are resistant to inhibition by most natural trypsin inhibitors and display some affinity for heparin. The existence of tryptase isozymes has been implied from the cloning of two tryptase cDNAs from human lung tissue, but distinct isozymes have not been isolated and characterized. This knowledge gap has been filled by isolating and characterizing two electrophoretically different forms of human lung mast cell tryptase, designated high-HLT (high molecular weight HLT) and low-HLT (low molecular weight HLT). These two forms of HLT have been separated by chromatography on a cellulose phosphate column, with the high M$\sb{\rm r}$ form eluting with 10 $\mu$M heparin and the low M$\sb{\rm r}$ form subsequently eluting with 1 M NaCl. Using HMWK and VIP as substrates, these two forms of HLT were found to differ with regard to specificity and rate of cleavage. High-HLT initially cleaved HMWK at a single Arg residue, whereas low-HLT cleaved HMWK simultaneously at multiple sites. Both isozymes cleaved VIP at multiple sites, but differed with regard to the preferential site of cleavage. Low-HLT was, on an active site basis, 25 and 2 times more active than high-HLT on HMWK and VIP, respectively. In addition, gel filtration of the isozymes yielded M$\sb{\rm r}$s of 125 Kda for high-HLT and 28 kDa for low-HLT, indicating tetrameric and monomeric quaternary structures, respectively. Both isozymes were inhibited by human secretory leukocyte proteinase inhibitor (SLPI), but not by other trypsin inhibitors tested. This work provides the first evidence for the existence of distinct tryptase isozymes, with supposedly different in vivo functions, and identification of an inhibitor that may control tryptase activity in vivo.

Thesis (Ph. D.)--University of Oregon, 2003. / Typescript. Includes vita and abstract. Includes bibliographical references (leaves 137-145). Also available for download via the World Wide Web; free to University of Oregon users.

Previous work in our laboratory indicates that the diameter of brain arteries and arterioles can be increased by N-methyl-D-aspartate (NMDA) receptor activation. We looked for expression of NMDA receptors and endothelial cell responses to NMDA receptor agonists and antagonists in the mouse brain endothelial cell line bEnd.3. Using RT-PCR and Western blotting we found evidence supporting the presence of NMDA receptor subunits NR1 and NR2C. Treatment of bEnd.3 cells with combinations of 100 μM glutamate and D-serine significantly increased intracellular calcium. However, we saw no direct evidence that NO was produced in response to NMDA receptor activation using the Griess method. We did observe an NMDA receptor-dependent increase in protein nitrosylation. This increase is unlikely related to enhanced NO levels since it was not correlated with NO production and was not inhibited by the endothelial NO synthase inhibitor L-NIO.