Signal transducer and activator of transcription (STAT) is a family of intracellular proteins that are responsible for carrying the signal from the cell surface to the nucleus in response to specific ligands. Once in the nucleus, STATs activate the transcription of specific genes. To date, seven human STATs have been identified. Among these STATs, STAT3 is considered as oncogenic. It activates genes that block apoptosis and inhibits antitumor immune responses (1). STAT3 is also essential in early embryogenesis and plays a role in cell growth and survival, differentiation and apoptosis depending on the target tissue. Analysing STAT3 signalling provides insights into pathology and can be used as a tool for diagnosis, prognosis and therapy development. Traditionally, western blot has been used to analyse cell signalling but it is impractical in analysing rare cell populations or providing information at the single cell level. Moreover, it is a demanding and time consuming technique that offers qualitative and less sensitive analysis. The rapid evolution in the multi-parametric flow cytometry and the availability of both epitope specific antibodies and sophisticated software facilitate the wide application of this technology in cell signalling studies. Flow cytometry has the ability to resolve different subcellular sets in a heterogeneous population, collects data at a single cell level and correlates multiple markers simultaneously. However, it requires highly standardized protocols for maximal sensitivity. The aim of this study was to assess the dose and the time response of both total STAT3 and pSTAT3 to in vitro stimulation with either IL-6 or IL-10 in peripheral blood mononuclear cells (PBMC). This assessment was done using both the flow cytometry and the western blot techniques. The results of this study showed that lower doses of IL-6 (1 & 10 ng/ml) were not sufficient to induce phosphorylation of STAT3. However, following stimulation with 100 ng/ml of IL-6, no significant change in the level of total STAT3 could be detected in either lymphocytes or monocytes from 3 different donors using either the FC500 or the Accuri cytometer. Using the FC500 cytometer, a small but insignificant increase in the pSTAT3 was seen in the lymphocytes and monocytes. A significant increase in STAT3 phosphorylation was only observed for monocytes after 15 minutes stimulation with 100 ng/ml of IL-6 using the Accuri flow cytometer. xii When the fluorescent labelled antibodies used in the flow cytometric assays were used for western blot probing, western blot analysis of stimulated cell lysates with 100 ng/ml IL-6 detects proteins of a low molecular weight than STAT3 or pSTAT3 which may explain the flow cytometric results of IL-6 stimulation. In IL-10 stimulation experiments, lower doses (1 and 10 ng/ml) tested by flow cytometric and western blot techniques demonstrated insignificant STAT3 phosphorylation induction. Following stimulation with either 50 or 100 ng/ml IL-10, no significant change in the total STAT3 was seen in either lymphocytes or monocytes when using the Accuri flow cytometer. However, stimulation with 100 ng/ml IL-10 induces STAT3 phosphorylation from 10 minutes through 30 minutes in both lymphocytes and monocytes. Longer times were required and high inter-individual variability was noticed for the activation of STAT3 after stimulation with 50 ng/ml IL-10. By using different antibodies from those used in the flow cytometric assay; the western blot results were comparable with the flow cytometric findings following stimulation with 100 ng/ml IL-10. The addition of phosphatase inhibitors during the flow cytometric protocol didn’t show any increase in the STAT3 phosphorylation. However, using paraformaldehyde for fixation and methanol for permeabilisation significantly decreased the mean fluorescence intensity of the PE conjugated antibodies comparing to the BD commercial fixation and permeabilisation buffers. The onset and the signal intensity of “in house” chemiluminescence mixture for western blot detection of STAT3 were comparable to the commercial ECL reagent used. However, the background of the “in house” mixture increased with time and was higher than with the commercial product. Upon longer exposure, the background increased enough to cause signal loss. In spite of the number of advantages of the flow cytometric assay compared to the western blot assay, these results are highly dependent on the specificity and the selectivity of the used antibodies. Furthermore, flow cytometry requires a highly standardized protocol to be able to assess the normal level of signalling proteins which could be later applied to detect abnormalities. It is suggested that the antibodies used in the flow cytometric assay be tested by western blot to confirm their selective detection of the target protein before their use in the flow cytometric analysis. / Dissertation (MSc)--University of Pretoria, 2013. / gm2014 / Pharmacology / unrestricted

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第18677号 / 医博第3949号 / 新制||医||1007(附属図書館) / 31610 / 京都大学大学院医学研究科医学専攻 / (主査)教授 髙折 晃史, 教授 長田 重一, 教授 河本 宏 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Brucella abortus is a facultative intracellular bacterial pathogen, which causes abortion in cattle and undulant fever in human. B. abortus strain RB51 (Strain RB51) is the official vaccine for bovine brucellosis in the USA. B. abortus strain RB51 can be used as a vector for the over-expression of its own (homologous) as well as heterologous protective antigens. The immune system can detect these heterologous antigens and produce a response. Expressing a protein in different bacterial compartments has been shown to affect its accessibility to the immune system and the way the antigen is processed by antigen presenting cells. In order to determine if the immune response is affected by the localization of the antigen, green fluorescent protein (GFP) was expressed at three different locations in B. abortus strain RB51, outer-membrane (OM), periplasmic space (PS) and in the cytoplasmic region (CR) of B. abortus strain RB51. This localization was obtained by transforming strain RB51 with plasmids pBBg18sGFP and pBBgSsGFP, in which the 18 kDa Brucella lipoprotein and the Brucella Cu/Zn SOD protein signal sequences were added to the GFP sequence to cause OM and PS expression respectively. No signal sequences were added to the plasmid pBBgGFP for CR only expression. Expression and localization of GFP in the different compartments in recombinant B. abortus strain RB51 were confirmed by electron microscopy and antibody absorption experiments. Groups of 5 female BALB/c mice each were injected and boosted with three recombinant strains and appropriate controls. Mice were bled and their anti-GFP antibody production was assessed. None of the immunized mice produced specific antibodies against GFP, probably due to the low expression of the heterologous antigen observed in this study by strain RB51 observed in this study. It will be necessary to produce new recombinants which are able to express higher amounts of GFP to answer if localization of heterologous antigen within the recombinant RB51 affects the level of a specific immune response. / Master of Science

DNA vaccines in larger animals, such as horses, are generally less effective and elicit significantly weaker immune responses, than in small animal model systems. To provide optimal protection against pathogenic microorganisms, the induction of both humoral and cellular immune responses from DNA vaccination may be necessary. One limitation to DNA immunization in the horse is the difficulty in generating high levels of antigen-specific antibody and CTL responses. Previous work in the laboratory has demonstrated that expression constructs containing native sequences encoding the surface unit (SU) envelope glycoprotein (pCiSU) of the Equine Infectious Anemia Virus (EIAV) are ineffective at stimulating immune responses in the horse. This was attributed to an unusual codon-usage bias of the EIAV genome that significantly limits the expression of SU sequences. Optimizing the codon usage of pCiSU (pSYNSU) in DNA vaccines stimulated low-titer immune responses in inoculated ponies. Another plausible explanation for the reduced effectiveness of these DNA vaccines may be transfection deficiency and low level expression elicited by plasmid vectors in the horse. These studies investigated if the addition of a cationic polymer, deacylated polyethyleneimine (PEI), and/or codon optimized molecular immune-stimulatory cytokines could augment the relatively weak immunogenicity of pSYNSU in DNA vaccination of horses/ponies. Administration of DNA in formulation with PEI resulted in the robust production of very long-lived humoral (15 months after vaccination) responses and induced cell-mediated IFN-y responses five days after immunization.. Additionally, the co-expression of a family of IL-15 cytokines expanded the repertoire of T cell recognition to SU-specific peptides, in terms of lymphoproliferation. DNA vaccination incorporating one IL-15 family member, IL-15 (SSLSS) significantly enhanced serum antibody levels of IgGA and IFN-y mRNA expression levels. These responses were distinctly different from results seen with vaccinates that received „naked‟ pSYNSU DNA vaccines. It is evident from these vaccine studies that PEI can enhance DNA vaccine-elicited antibody and CTL-associated responses in the horse and IL-15 (SSLSS) can dramatically augment these responses. These results demonstrate an important role for PEI in promoting the longevity of immune responses to genetic immunization, which has not been reported previously in any large animal model.

A model for the study of the relationship of immunity to cancer is found in AKR mice which harbor Gross virus. This genetically transmitted virus is present in a latent form for months before it spontaneously induces leukemia. Many investigators have demonstrated near normal humoral responses, but abnormal cellular immunity in the preleukemic animal. With increasing age, pathology of the disease is expressed, reflecting diminished immunity. In this study, the ontogeny of humoral antibodies of AKR/J and SWR/J mice was assayed by microagglutination techniques in response to thymus-independent, thymus-dependent, and solubilized antigens. Simultaneous injections of thymusdependent and -independent antigens provided data suggesting an impaired humoral response in the AKR mouse.

Understanding how organisms function is central to Biology. Assessing how animals respond to fluctuations in their environment and determining inter-individual variation in phenotypic plasticity is paramount to identifying the physiology of traits, the selective pressures which have shaped them, and how we can manipulate them to benefit human life. The over-arching goal of my thesis is to understand the effects of sex, diet and pathogen exposure on the physiology of the fruit fly to assess the versatility of their individual traits in response to these natural factors. Chapter 2 investigates how the sexes utilise nutrition towards their lifespan and reproduction, providing evidence that the reproduction of males and females requires different dietary components while lifespan does not. Chapter 3 reveals that the sexes also differ in how they utilise nutrients for pathogen resistance identifying that females are highly protein-limited and more susceptible to infection than males. Chapter 4 provides the first comprehensive study of how organisms alter their dietary intake in response to infection, finding that flies behaviourally ingest less and consume higher protein:carbohydrate ratio diets when exposed to live fungal spores. Chapter 5 explores the phenomenon of trait-enhnacing external stresses, a response often termed hormesis. This study reveals that the beneficial physiological response from inactive fungal spore exposure, a potential form of hormesis, incurs immune costs. The implications of my results to the field of physiology are discussed in Chapter 6 where I also highlight the limitations of my work and potential consequences for life history research. Overall it is determined that studies investigating the natural physiological response of organisms or potentially beneficial treatments for our own species, must consider sex-specific effects, physiological consequences in a variety of traits, and how organisms may utilise variation within their environment to alter their phenotypic condition.

Many parasites have evolved to release products that inhibit host defence mechanisms such as enzymes in the mammalian host, in order to promote and sustain their survival within the host. The human filarial nematode Brugia malayi produces larval microfilariae, which circulate in the blood stream. Their most abundant secreted product is a serine protease inhibitor Bm-SPN-2. Serine protease inhibitors (Serpins) are reported to be involved in how the nematodes avoid host immune defences, and in the case of Bm-SPN-2, the protein was found to specifically inhibit the enzymatic activity of human neutrophil elastase and cathepsin G in a dose-dependent manner. More recently, these two enzymes have been linked to the activation of a major innate cytokine IL-33, which is stored as a full-length 270-aa protein in the cell nucleus, and released as an active C-terminal domain upon stimulation. As full-length (FL) human and murine IL-33 are not commercially available, soluble murine and human FL-IL-33 were produced in transfected HEK 293T cells, following mutation of the nuclear binding motif. In this form, IL-33 is no longer retained in the nucleus and can be purified as a soluble protein. It was confirmed that once cleaved, recombinant human IL-33 was able to induce significant IL-6 secretion by mast cells. Bm-SPN-2 was then shown to block human full-length IL-33 cleavage by inhibiting human neutrophil cathepsin G in a dose dependent manner, supporting the hypothesis that Bm-SPN-2 may act in vivo to prevent IL-33 activation and the promotion of the TH2 immune response. However, in the in vivo setting, it was unexpectedly found that IL-33R (ST2) gene deficiency did not enhance the survival of B. pahangi microfilariae. Furthermore, in the absence of IL-33R, murine immune responses to microfilariae were not significantly altered compared to wild-type BALB/c mice, other than in a significant increase in IL-33 expression. Hence while Bm-SPN-2 can act in vitro to forestall one of the key events in TH2 induction, this has not yet been shown to be crucial to the immune response to the parasite in vivo.

Parasites have a major impact on host condition and fitness and thereby represent a strong selective force for individuals in wild populations. The main defence against parasite infection and associated morbidity is the host immune response, and consequently it is expected for there to be strong selection eroding genetic variation underlying immune responses in natural populations. However, studies in the wild have found considerable heritable variation underlying immune responses. Few studies have investigated the genetic variants underlying immunity in wild populations and are able to examine how genetic variation is maintained in the face of natural selection. The aim of this thesis is to investigate the selection on, and genetic variation underlying, immunity in a wild Soay sheep population by looking at antibody responses to the prevalent parasite Teladorsagia circumcincta. Anti- T. circumcincta antibody levels (IgA, IgE, IgG) were measured in neonatal plasma samples taken soon after birth, representing maternally-derived antibodies, and in samples from August yearly from four month old lambs and adults, representing endogenous antibodies. All three endogenously produced antibody measures in lambs and adults were repeatable and heritable. In addition, a genome wide association study run on the three antibody traits on August lamb and adult measures found associations between anti-T. circumcincta IgA levels and single nucleotide polymorphisms in a region on chromosome 24. There was evidence for age- and isotype- dependent negative associations between antibody isotypes and strongyle faecal egg counts (FEC). Further, there was evidence for age-dependent selection via positive associations between anti-T. circumcincta IgG and survival in females and annual fecundity in males. In comparison, there was no additive genetic variance underlying maternally-derived (neonatal) anti-T. circumcincta antibody levels in neonates, but maternal and maternal genetic effects explained a considerable proportion of the variance in these traits. There was evidence for associations between neonatal anti-T. circumcincta IgG and later offspring phenotype and fitness, independent of total antibody (IgG) transferred. We found that neonatal anti-T. circumcincta IgG levels positively predicted survival to four months old, as well as weight in August. In addition, neonatal anti-T. circumcincta IgG levels were associated with reduced strongyle FEC in August, and were associated with improved survival over the first winter. In early life, maternally-derived anti-helminth antibodies are important for early growth, survival, and parasite resistance, as well as first winter survival, while fitness benefits in adulthood were associated with higher endogenous anti-helminth antibody levels. This thesis illustrates that maternal effects and genetic variation can have strong effects on variation in immunity in the wild, and this variation in turn can have health and fitness consequences for individuals.

Background The prevalence of atopic disease among children in the formerly socialist countries in Europe, with a life style similar to that prevailing in Western Europe 30–40 years ago, is low, whereas there has been a pronounced increase in industrialized countries over the last decades. The environment during infancy influences the risk of developing allergy for many years, perhaps even for life. Objective To investigate the development of allergen-specific cytokine responses during the first 2 years of life in two geographically adjacent countries with marked differences in living conditions and incidence of atopic diseases, i.e. Estonia and Sweden. Methods The development of immune responses to food (β-lactoglobulin (BLG) and ovalbumin (OVA)) and inhalant (cat and birch) allergens was studied from birth up to the age of 2 years in 30 Estonian and 76 Swedish infants. Clinical investigation and skin prick tests were performed and blood samples were obtained at birth and at 3, 6, 12 and 24 months. Results The levels of IL-5, IL-10 and IL-13 secreted by peripheral blood mononuclear cells stimulated with BLG, OVA and cat allergen in Estonian and Swedish infants declined during the first 3 months of life. All cytokines then progressively increased in the Swedish infants, indicating the replacement of non-specifically responding immature cord blood T cells with specific T memory cells, which are primed postnatally. The resurgence of allergen-specific responses in the Estonian infants was less marked. These differences were particularly notable for birch-specific T cell responses, which correlated with development of atopic disease in the Swedish children. Conclusions The development of specific T cell memory to food and inhalant allergens during the first 2 years of life differs between infants living in Sweden and Estonia, and mirrors the disparate patterns of expression of allergic disease which subsequently develops in the respective populations.

Essential fatty acids nutrition and its effects on immune responses of the juvenile grouper, Epinephelus malabaricus Feng-Cheng Wu (Advisor: Dr. Houng-Yung Chen) Institute of Marine Biology, National Sun Yat-sen University Kaohsiung 804 Taiwan. A series of three experiments was conducted to study the essential fatty acids nutrition and its effects on immune responses (IR) of the juvenile grouper, Epinephelus malabaricus. All experimental diet contained 10 g/100 g diet supplemental lipids from various sources. A reference diet was used in all experiments and contained natural oil mixture of cod liver oil, linseed oil, and safflower oil at a rate of 2:1:1 (wt/wt/wt). In experiment 1, juvenile grouper (11.8 ¡Ó 0.7 g) were fed for 12 wks on one of the seven experimental diets, control diet and the reference diet to investigate dietary requirement for docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) and effects on IR of grouper. Seven experimental diets contained 1 g/100 g diet of DHA and EPA in various combinations and 9 g/100 g diet of tristearin. The control diet contained 1 g/100 g diet of trilinolenin and trilinolein (3:1, wt/wt). The results indicate that there was a significant difference among dietary treatments in growth, phagocytosis and leucocytes proliferation, when stimulated by Con A and PHA-P but not by LPS. However there was no difference in survival rate and relative liver weight. Enhanced growth was observed when the dietary DHA/EPA was greater than 1, indicating that DHA was superior to EPA in promoting fish growth. DHA is the only member in the family of (n-3) highly unsaturated fatty acid (HUFA) that stimulates phagocytic functions of leucocytes and T cell proliferation of the juvenile grouper. In experiment 2, juvenile grouper (11.3 ¡Ó 0.6 g) were fed for 12 wks on one of the eight experimental diets or the reference diet to investigate dietary requirement for linolenic acid (LNA) and linoleic acid (LA), as well as effects on nonspecific IR of grouper. The test diets were supplemented with LNA or LA at a rate of 1 or 2 g LNA or LA/100 g diet or 2 g/100 g diet of LNA and LA in various ratios (3, 1.4, 0.7 and 0.4). Tristearin was used to fill the lipid supplemental level to 10 g/100 g diet. The results show that enhanced growth and optimal non-specific cellular IR were observed when the grouper were fed on the diet having the highest LNA/LA ratio (3:1) or on the diets supplemented with LNA. But the enhancement was not different (P>0.05) from that of the reference diet group. Thus, incorporating 2 g/100 g LNA/LA (3:1, wt/wt) in diet ensures adequacy of the grouper for essential fatty acid. In experiment 3, juvenile grouper (13.2 ¡Ó 0.9 g) were fed on one of the six experimental diets in the 2 ¡Ñ 3 factorial design or on the reference diet for 12 wks to investigate dietary requirement for (n-3)HUFAs and arachidonic acid (AA), as well as effects on IR of grouper. Two levels of (n-3)HUFAs (1 or 2 g/100 g ) in combination with 3 levels of AA (0, 1 or 2 g/100 g) were tested. The results show an enhanced growth when optimal concentrations of AA and (n-3)HUFA were incorporated to the diets. Liver (n-6)HUFAs concentration reflects IR of the juvenile grouper. Interaction of AA and (n-3)HUFAs in affecting fish growth and IR was insignificant (P>0.05), and concentrations of dietary (n-3)HUFAs or AA did not significantly affect fish survival rate. The results of the 3 experiments show that the grouper will benefit most in growth and IR when their diets contain 1 g/100 g diet of (n-3)HUFA and 1 g/100 g diet of AA, when (n-3)HUFA is a mixture of DHA and EPA at a ratio of 3:1 (wt/wt).