Thesis (Ph.D. (Chemical Pathology))--University of the Witwatersrand, Faculty of Health Sciences, 2012 / Alkaline phosphatases (ALPs) are a group of membrane-bound glycoproteins that occur in many species of animals and have a wide tissue distribution. ALPs have been shown to play a role in cell differentiation and organogenesis. In humans, the physiological role of ALP in skeletal mineralization is well documented. In routine clinical practice, ALP measurement is frequently used in the differential diagnosis of liver and bone diseases. Studies have shown the presence of tissue non-specific ALP (TNSALP) activity in rat adipocytes, human preadipocytes and in a murine preadipocytic cell line, 3T3-L1. ALP has also been shown to play a role in adipogenesis in 3T3-L1 cells and human preadipocytes. The purpose of the present study was to determine whether the ALP that is expressed in a human hepatocarcinoma cell line, HepG2 has a role in intracellular lipid accumulation. Intracellular lipid droplet accumulation in HepG2 cells was induced by addition of oleic acid coupled to albumin (Sigma-Aldrich, UK) to culture medium (Earle’s Minimum Essential Medium [EMEM]) and used at a final concentration of 400M. Tissue non-specific ALP inhibitors (levamisole and histidine) inhibited ALP activity and intracellular lipid accumulation in both the 3T3-L1 and HepG2 cells. Post-transcriptional silencing of the tissue non-specific alkaline phosphatase (TNSALP) gene using siRNA oligos inhibited intracellular lipid accumulation in both 3T3-L1 and HepG2 cells. In both cell lines, the ALP mRNA levels decreased in cells transfected with the anti-ALP siRNA compared to untransfected cells. This decrease in gene expression was mirrored by a corresponding fall in ALP activity in both cell lines. Quantification of the expression levels of the peroxisome proliferator activated receptor gamma (PPAR) gene (an important regulator of adipogenesis) using real-time quantitative polymerase chain reaction (real-time qPCR) showed an upward regulation of its expression four days after induction of intracellular lipid droplet accumulation in both cell types after which the levels declined. Neither levamisole nor histidine affected the expression of PPAR. Immunostaining of HepG2 cells with monoclonal antibodies against adipophilin and staining for ALP using the ELF 97 kit (Molecular Probes, Holland) demonstrated that ALP activity was localized to the surface of the lipid droplet membrane. A previous investigation has shown that ALP activity is higher in preadipocytes isolated from black compared to white females. Investigation of single nucleotide polymorphisms in the promoter region of the human TNSALP gene shows that genetic variation in the ALP promoter is not responsible for the ethnic differences in ALP activity observed in black and white South Africans. In conclusion, the close association of ALP activity with the lipid droplet membrane in HepG2 and 3T3-L1 cells and the ability to block intracellular lipid accumulation using sequence specific oligonucleotides for ALP and pharmacological agents (histidine & levamisole) strongly indicates that ALP is involved in intracellular lipid accumulation in HepG2 cells and 3T3-L1 cells. This study also shows that PPAR gene expression increases during lipid accumulation in HepG2 cells but that inhibition of ALP with histidine or levamisole does not affect the expression of this gene. Thus, ALP must act downstream of PPAR during intra-cellular lipid accumulation

Thesis. / Includes bibliographical references (p. 27-39).

Thesis. / Includes bibliographical references (p. 27-39).

Th.--Pharm.--Montpellier 1, 1978.

This dissertation describes our investigation into the regulation and function of the innate immune modulator alkaline phosphatase (ALP). Animal intestine harbors a vast and complex microbial community, the gut microbiota. While resident microbes perform vital functions and confer tremendous benefits on their hosts, they also provide antigens and toxins that provoke host immune responses, which, if uncontrolled, could be detrimental to the host. Our lab previously demonstrated a negative feedback loop mediated by intestinal ALP (ALPI) which promotes immune tolerance to the commensal microbiota in zebrafish. We continue to investigate regulation mechanisms of ALP genes and explore their roles in modulating host-microbe interactions in various models. We have characterized four zebrafish alp genes, and we engineered tools for functional studies of these genes. Phylogenic analyses involving zebrafish alp genes revealed distinct evolution histories of animal ALP genes and implied their diversified functions. We then tested whether the regulation mechanism and the roles of zebrafish alpi were conserved in mice. We found the ALPI gene Akp3 was specifically upregulated by microbiota and played a role in immune education. We demonstrated the contribution of innate immune signaling to animal weight gain induced by high fat diet feeding. Finally, we discovered the positive correlation between neonatal ALPI activity and gestational age, suggesting potential therapeutic value of ALP supplementation for preventing necrotizing enterocolitis development in preterm infants. This dissertation includes previously published and unpublished co-authored material.

The development of alkaline phosphatase (AP) has been studied in two species of Urodele amphibian, Ambystoma qracile and Taricha torosa. The enzyme is present in embryo homogenates at gastrulation and increases immensely in activity as development proceeds to the free-swimming stages. The activity level is a product of two isozymic forms that change quantitatively. Using histochemical detection methods, it was possible to correlate the specific activity and electrophoretic data with histological AP development. Some function of AP were related to the available data. A correlation between substrate specificities and function is proposed which may assist in understanding the role of AP in the process of differentiation / Science, Faculty of / Zoology, Department of / Graduate

Clostridium difficile est un bacille à gram-positif anaérobie strict et sporulant. Cet entéropathogène est responsable de diarrhées post-antibiotiques et de colites pseudomembraneuses. Après germination des spores dans le petit intestin, les cellules végétatives de C. difficile vont être soumises à de nombreux stress comme des peptides antimicrobiens, des variations d’osmolarité et de pH ainsi qu’une faible tension d’O2 à proximité des cellules épithéliales du colon. De plus, la production des toxines va favoriser une forte inflammation responsable de la production de nombreux composés oxydants tels que les espèces réactives de l’oxygène (ERO), du NO et des espèces réactives de l’azote (ERN). La présence de ces différents stress suggère que C. difficile possède des mécanismes de détection, de protection et de détoxification. Chez les firmicutes, plusieurs de ces mécanismes sont contrôlés par le facteur sigma alternatif impliqué dans la réponse générale aux stress, SigB. De manière intéressante des gènes codant SigB et ses régulateurs (RsbV et RsbW) sont présents dans le génome de C. difficile, cependant aucunes données concernant l’existence ou le rôle d’une réponse générale aux stress ne sont présentes chez cette bactérie. Nous avons dans un premier temps construit un mutant sigB et nous avons montré que cette inactivation n’entraînait pas de défaut de croissance. Via une analyse globale de l’expression des gènes, nous avons montré que SigB contrôle 20% des gènes de C. difficile à l’entrée en phase stationnaire. SigB contrôle négativement la sporulation. SigB contrôle positivement plusieurs gènes codant des protéines associées à la surface et pouvant intervenir dans le processus d’adhésion aux cellules de l’hôte. SigB joue également un rôle crucial dans l’adaptation aux stress rencontrés lors de l’infection dans le tube digestif et dans la colonisation. En effet, un mutant sigB est plus sensible aux pH légèrement acides, à certains peptides antimicrobiens, aux ERO, à différents composés générant du NO ainsi qu’aux faibles tolérances en O2. L’étude de la voie de signalisation aboutissant à l’activation de SigB a également été étudiée au cours de ce projet de thèse. Le gène sigB est le dernier gène d’un opéron constitué de CD0007 et CD0008, codant des protéines aux fonctions inconnues, ainsi que de rsbV et rsbW, codant respectivement le facteur anti-anti-sigma et le facteur anti-sigma. De manière intéressante, l’expression de sigB ne semble pas autorégulée chez C. difficile, à l’inverse des autres firmicutes. Les interactions entre RsbV et RsbW ainsi que RsbW et SigB, impliquées dans l’activation de SigB sont présentes chez C. difficile. La délétion du gène rsbV entraîne une augmentation de la sensibilité aux composés générant du NO et une diminution de la tolérance à l’O2. Ces résultats sont en accord avec la diminution de l’expression des gènes cibles de SigB chez le mutant rsbV. La phosphatase impliquée dans le processus d’activation de SigB, en déphosphorylant vraisemblablement RsbV, a également été identifiée et inactivée. La délétion du gène codant cette phosphatase, CD2685 conduit à l’augmentation de la sensibilité aux composés générant du NO ainsi qu’à la diminution de la tolérance à l’O2, en accord avec son implication dans la voie d’activation deσB. L’activité de SigB augmente suite à une carence nutritive. En effet, l’expression des gènes cibles de SigB est induite de manière dépendante de SigB et de RsZ à l’entrée en phase stationnaire ou après exposition au CCCP, un composé réduisant le niveau intracellulaire d’ATP. Ces travaux de thèse permettent d’établir un rôle prépondérant de SigB et de sa voie d’activation au cours de l’infection notamment dans la protection et la détoxification de stress majeurs que C. difficile est susceptible de rencontrer dans le tube digestif / Clostridium difficile is a tram-positive, anaerobic, spore-forcing bacterium. This enteropathogen is a major cause of antibiotic-associated diarrhea and of pseudomembranous colitis, a potentially lethal disease. After germination, vegetative cells encounter different stresses such as pH variations and hyperosmolarity as well as antimicrobial peptides. Moreover, the production of toxins triggers an important inflammation process leading to the production of several antimicrobial compounds such as reactive oxygen species (ROS), nitric oxide (NO) and reactive nitrogen species. The presence of these different stresses suggests that C. difficile has developed some mechanisms of detection, protection and detoxification. In the firmicutes, several of these mechanisms are controlled by the alternative sigma factor involved in the general stress response, SigB. Interestingly, genes encoding SigB and its regulators (RsbV and RsbW) are present in the genome of C. difficile, however less is known about the role of a general stress response in this bacterium. We constructed a sigB mutant and we showed that sigB inactivation does not lead to a growth defect. Using a transcriptomic analysis, we showed that SigB controls around 20% of the genes of C. difficile at the onset of the stationary phase. SigB negatively controls the sporulation process. SigB positively controls several genes encoding surface associated proteins likely involved in the adhesion to the host cells. SigB plays a crucial role in the stress management including several stresses C. difficile likely encounter during its infectious cycle. Indeed, the sigB mutant is more sentsitive to low pH, to some antimicrobial compounds, to ROS, to several NO-donor compounds as well as to low oxygen tension. The signaling pathway involved in the activation of SigB has been studied in the PhD project. The sigB gene is the last gene of an operon in which belong CD0007 and CD0008, encoding town unknown function proteins, as well as rsbV and rsbW, encoding the anti-anti-sigma factor and the anti-sigma factor of SigB, respectively. Interestingly, contrary to the other firmicutes, the expression of sigB does not seem to be auto-regulated. Protein interactions between RsbV and RsbW as well as RsbW and SigB, involved in the activation of SigB, are present in C. difficile. The disruption of rsbV leads to a higher sensitivity to NO-donor compounds as well as low oxygen tension. These results are in agreement with the decreased expression of several SigB target genes in the rsbV mutant. The phosphatase involved in the activation of SigB, likely by dephosphorylating RsbV, has been identified and disrupted. The disruption of CD2685, encoding this phosphatase, leads to a higher sensitivity to NO-donor compounds as well as a lower tolerance to low oxygen tension, in agreement with its involvement in the SigB activation pathway. The activity of SigB increased after an energetic starvation. Indeed, the expression of SigB target genes are increased in a SigB and CD2685 dependent manner at the onset of the stationary phase or after CCCP exposure, a compound decreasing the intracellular level of ATP. This work allows to show a crucial role of SigB and its activation pathway during the infection notably in the protection and the detoxification of the major stresses C. difficile is likely to encounter in the gut

Die leukozytäre alkalische Phosphatase (leukoAP) wird in der Humanmedizin zur Charakteristik stabkerniger neutrophiler Granulozyten und damit der Feststellung leukozytärer Reaktionen im Sinne der Linksverschiebung genutzt. Des weiteren dient die quantitative Erfassung der leukoAP der Funktionsbeschreibung dieser Zellart. Im Vergleich dazu liegen bisher beim Rind nur wenige Untersuchungen vor. Ziel dieser Arbeit war es deshalb, entsprechende Grundlagen für diese Tierart zu erarbeiten, d.h., die Bestimmungsmethodik an Rindergranulozyten zu adaptieren, Referenzwerte zu definieren sowie klinische Bezüge bei ausgewählten Krankheiten zu prüfen. Die Untersuchungen wurden an gesunden weiblichen Kälbern im Alter von drei bis vier (n = 13), sechs bis acht (n = 13) und 12 bis 14 Wochen (n = 11), weiblichen Jugrindern im Alter von sechs (n = 9) und zwölf Monaten (n = 10) sowie dreijährigen Kühen in der Trockenstehperiode (n = 11), fünf bis sieben Wochen (n = 10) sowie 12 bis 14 Wochen post partum (n = 6) durchgeführt. Außerdem wurden Kühe mit Dislocatio abomasi (n = 8) und mit weiteren Erkrankungen (n = 8) analysiert. Zur Analytik der leukoAP wurde ein durchflußzytometrisches Verfahren für Rinder adaptiert, das eine schnellere und genauere Arbeit als das herkömmliche Verfahren der leukoAP-Index-Bestimmung ermöglicht. Weiterhin wurden die Aktivitäten der AP und dessen Knochenisoenzyms im Blutserum sowie das Leukogramm bestimmt. Die Bestimmung der leukoAP ist auf der Basis der Diazoniumfärbung auch beim Rind durchflußzytometrisch mit großer Präzision und hohem Probendurchsatz möglich. Die leukoAP-Aktivitäten (relative Fluoreszenzaktivitäten) zeigen eine Altersabhängigkeit. Sie bewegen sich bei drei bis vier Wochen alten gesunden Kälbern zwischen 2,2 bis 5,7 (Mittelwert: 3,8), sinken kontinuierlich bis in den Bereich 1,9 bis 4,3 (Mittelwert: 2,8) bei 12 bis 14 Wochen alten Kälbern ab (p£0,05), um bis auf 2,6 bis 5,5 (Mittelwert: 4,2) bei einjährigen Jungrindern wieder signifikant (p£0,05) anzusteigen. Bei gesunden, trockenstehenden dreijährigen Kühen sowie Kühen 12 bis 14 Wochen post partum schwanken die leukoAP-Aktivitäten zwischen 1,2 bis 9,7 (Mittelwerte bei 3,3 bzw. 3,6). Im Zeitraum fünf bis sieben Wochen post partum ist die leukoAP-Aktivität mit 2,1 bis 8,2 (Mittelwert: 5,3) signifikant (p£0,05) höher. Bei der Gesamtheit der untersuchten gesunden Rinder korreliert die leukoAP weder mit der Zahl der Leukozyten (gesamt), der stabkernigen neutrophilen Granulozyten, der segmentkernigen neutrophilen Granulozyten, der Monozyten, noch mit der Zahl der Lymphozyten systematisch gesichert. Andererseits sind Beziehungen zu diesen Parametern für einzelne Alters- und Laktationsgruppen nachweisbar. Die leukoAP korreliert weder mit der Gesamtaktivität noch mit dem Knochenisoenzym der AP im Serum. Kühe haben einen Tag nach Reposition einer Dislocatio abomasi leukoAP-Aktivitäten von 2,4 bis 7,4 (1. und 3. Quartil). Innerhalb von drei Tagen post repositionem steigt die leukoAP-Aktivität in den Bereich 5,1 bis 11,5 signifikant an, während die Leukozytenzahl sinkt (p£0,05). Bei Kühen mit linksseitiger Dislocatio abomasi verlaufen die leukoAP-Aktivität sowie der Anteil stabkerniger neutrophiler Granulozyten postoperativ parallel. Bei Kühen mit entzündlichen Erkrankungen (Klauensohlengeschwüre, Retentio secundinarum, Pneumonie, Ekzem) sind z.T. stark erhöhte leukoAP-Aktivitäten feststellbar. Sie gehen tendenziell mit einem Anstieg der stabkernigen neutrophilen Granulozyten sowie mit Leukozytose einher, im Einzelfall besteht jedoch nicht immer dieser Trend. Die Bestimmung der leukoAP ist nur bei gegebenen technischen Voraussetzungen eine auch bei Rindern einfach durchführbare Methode zur Charakteristik der neutrophilen Granulozyten mit altersabhängigen Differenzen. Bei Dislocatio abomasi besteht eine Reduzierung der leukoAP-Aktivität, die post repositionem jedoch erheblich zunimmt. / Leukocyte alkaline phosphatase (leukoAP) is used in the human medicine to characterize unsegmented neutrophile granulocytes and thereby to diagnose leukocyte reactions in terms of a leftward shift. Furthermore, the quantitative capture of leukoAP serves as a functional description of this cell type. To our knowledge, only little investigation has been done on cattle. Therefore, this dissertation aimed to acquire an equal basis for this species, i.e. to adapt the methodology of determination, to define reference values, and to examine relevant relationship for selected diseases. Healthy female calves aged between three and four (n = 13), six and eight (n = 13), and between twelve and fourteen weeks (n = 11), cows aged six (n = 9) and twelve months (n = 10), three-year-old cows ante partum (n=11), and ten cows between five and seven as well as six cows between twelve and fourteen weeks post-partum were used in the study. In addition, eight cows with abomasal dislocation and another eight with other complications were examined. For analyzing leukoAP we adapted flow cytometry for cattle which delivers faster and more exact determination than the conventional method of leukoAP index determination. Moreover, the activity of alkaline phosphatase (AP) in blood serum and its iso-enzymes in the bone as well as leukogrammes were determined. The flow cytometrical determination of leukoAP activity was based upon the diazonium staining technique which also provides an accurate measurement in a large number of samples for cattle. The leukoAP activities (given as a relative fluorescence activity) showed age dependency: they ranged between 2.2 and 5.7 (mean value 3.8) in calves of 3 to 4 weeks in age, and decreased continuously and significantly (p£0,05) to 1.4 - 4.3 (mean value 2.8) in calves between 12 and 14 weeks of age, but increased again significantly (p£0,05) ranging between 2.6 - 5.5 (mean value 4.2) in one-year-old cattle. In healthy cows, ante partum and 12 to 14 weeks post-partum, differed the leukoAP activity between 1.2 and 9.7 (mean value 3.3 and 3.6, respectively), whereas in cows 5-7 weeks post-partum the leukoAP activity was significantly greater reaching 2.1 to 8.2 (mean value 5.3, p£0,05). For healthy cattle, leukoAP correlated neither with the total number of leukocytes, unsegmented neutrophile granulocytes, monocytes nor with the number of lymphocytes. In addition, leukoAP correlated neither total activity nor with serum AP and its iso-enzymes in the bone. One day after replacing the abomasal dislocation (between 1st and 3rd quartile) cows showed a significant decrease in leukoAP activity (2.4 - 7.4) vs. healthy cows after the same period of time post-partum. This parameter increased, however, significantly three days post-operative (5.1-11.5), whereas the number leukocytes decreased (p£0,05). In cows with left side abomasal dislocation, leukoAP activity and the number of unsegmented neutrophile granulocytes post-operative developed concomitantly. We found enhanced leukoAP activity in cows with different inflammatory disorders such as sole ulcer, placenta retention, pneumonia and eczema which provably (p£0,05) corresponded with an increased number of unsegmented neutrophile granulocytes and partly leukocytosis. However, for isolated cases this trend did not always show. Determining the leukoAP is, hence, a method for characterizing neutrophile granulocytes depending on age differences and is only practible for cattle if the required technical conditions are given. During abomasal dislocation, leukoAP activity decreased, but increased considerably after post-operative replacement.

The membrane bound alkaline phosphatase activity' in vegetative cells of Dictydstelium discdideum was found to exhibit a 8 to 10 fold increase in specific activity when incubated at 50°C. This activation was reversed on transfer of the preparation to 0°C. Similar activation of the vegetative enzyme was achieved by dialysing the crude membrane preparation, suggesting the removal of a low molecular weight inhibitor. Alkaline phosphatase solubilized from the membranes using Triton X-100 was similarly activated by 50°C treatment or dialysis and the 50°C activation was reversed by incubation at 0°C. Both the dialysed vegetative membrane and the dialysed Triton X-100 extract were inhibited by the addition of concentrated dialysate. This inhibition could be relieved by subsequent dialysis. A 620 fold purified alkaline phosphatase preparation was obtained from crude vegetative membranes by affinity and ion exchange chromatography after solubilization of the enzyme using Triton X-100-5'-nucleotidase activity copurified along with the alkaline phosphatase activity in all the fractionation steps employed. The membrane bound 5'-nucleotidase activity was not activated either by incubation at 50°C or by dialysis and the activity was far less stable than the alkaline phosphatase. However, the Triton X-100 extracted 5'-nucleotidase was activated by dialysis to the same extent as the alkaline phosphatase and both activities in the partially purified preparation were equally inhibited by the dialysates from vegetative membranes. These results suggest that both alkaline phosphatase and 5'-nucleotidase are due to a single protein but the interaction of the two substrates, AMP and pNPP, with the enzyme are different and are markedly influenced by conformational changes induced by the inhibitor. Unlike the vegetative enzyme, the alkaline phosphatase activity in the culminating membrane was not markedly activated by incubation at 50°C or by dialysis. Since the vegetative enzyme was activated by both treatments to levels similar to those found in culminating cells, it was proposed that the developmental increase in alkaline phosphatase in D.discoideum was due to the unmasking of already existing enzyme by the removal of inhibitor. However, the alkaline phosphatase activity of culminating cells differed from that of vegetative cells in chromatography on DEAE-Sephacel and conA-Sepharose and it was less stable in low concentrations of Tris-Cl and in SDS. In contrast, the culminating enzyme was more stable in high concentrations of Tris-Cl. These results suggest that the vegetative enzyme is modified during development. This modification appears to be slight, since the vegetative and culminating enzymes migrate identically in SDS polyacrylamide electrophoretic gels and the enzymes from the two developmental stages were similar in pH optima, in inhibition by phosphate and in inhibition by concentrated dialysate. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

published_or_final_version / Biological Sciences / Master / Master of Philosophy