Type 2 (Th2) immune responses are required for immune defence against helminths, but can also have pathogenic effects in allergic conditions. This thesis examined two factors which may influence Th2 immunity at a cellular and molecular level: cross-talk between Natural Killer (NK) cells and dendritic cells (DCs) and the cell surface organisation of DCs. Cross-talk between NK cells and DCs is well-established to impact Th1 responses against tumours and infection; however the influence of this interaction during Th2 inflammation is unknown. To investigate this, human monocyte-derived DCs were stimulated in vitro with different pathogen-associated molecules; LPS or Poly(I:C) which polarise a Th1 response, or soluble egg antigen (SEA) from the helminth worm Schistosoma mansoni, a potent Th2-inducing antigen. These cells were then combined with autologous NK cells. Confocal microscopy showed polarisation of the NK cell microtubule organising centre (MTOC) and accumulation of LFA-1 at contacts between NK cells and immature or Th2-polarising DCs, but not Th1-polarising DCs, indicative of the assembly of an activating immune synapse. NK cells did not lyse DCs treated with LPS or Poly(I:C), but degranulated to and lysed both immature DCs and Th2 polarising DCs. Antibody blockade of NK cell activating receptors NKp30 and DNAM-1 prevented this lysis. Furthermore, depletion of NK cells in mice which were then transferred with Th2 polarising DCs led to an enhanced Th2 recall response. Thus, these data indicate a previously unrecognised role of NK cell cytotoxicity in restricting the pool of DCs involved in Th2 immune responses. Secondly, this thesis investigated the nanoscale organisation of MHC-II on the surface of Th1 and Th2 polarising DCs using ground state depletion super-resolution microscopy. MHC-II was relatively homogenously distributed across the membrane with no significant changes in clustering between immature, Th1 and Th2 polarising DCs. In contrast, imaging CD74, which can mediate internalisation of MHC-II, revealed increased expression and a more homogenous distribution of this receptor on the surface of Th2-polarising DCs compared to Th1-polarising DCs. These data suggest that changes in the clustering of CD74 could modulate MHC-II surface expression during Th2 responses. Overall, the results in this thesis indicate that both molecular and cellular level modulation of DC function contribute to the development of Th2 responses.

Allergies are considered atopic diseases, or diseases that cause the immune system to create an abnormal amount of IgE antibodies when the body is exposed to an allergen. Allergies affect many people around the world, however many studies have shown a higher rate of allergy in developed countries when compared to developing countries. This discrepancy is hypothesized to be in part because of a decrease in parasitic infections, which have shown to have a protective effect for autoimmune-type diseases, like allergies. There are not many long-term, effective allergy treatments, however a promising allergen-specific immunotherapy technique uses a vaccine that targets B cell epitopes with the hope of increasing the amount of IgG antibodies as opposed to IgE specific antibodies to decrease the likelihood of an allergic reaction. This paper proposes a study that combines the protective effects of a parasite infection with a helminth infection and a B cell epitope vaccination, an already studied BM32 vaccine, to improve allergy symptoms of those with grass pollen allergy. This combination treatment will aim to decrease the number of symptomatic days, eosinophil count found at a scratch test site, and IgE antibodies found within the blood in grass pollen allergic people during peak grass pollen season.

Schistosomiasis continues to be an important cause of parasitic morbidity and mortality world-wide. Determining the molecular mechanisms regulating the development of schistosome-induced pathology, including granuloma formation, fibrosis and splenomegaly, is essential for understanding how schistosomes influence the immune system of the mammalian host. I report on the first whole genome microarray analysis of the murine liver and spleen during the progression of Schistosoma japonicum infection and of S. japonicum-Soluble Egg Antigen (SEA)-stimulated macrophages. My analyses of the infected liver revealed a distinct temporal relationship between the expression of chemokines and the recruitment of cells to the liver. T-cell and B-cell chemoattractants were up-regulated earlier reflecting the recruitment of these cells to the liver as illustrated by flow cytometry. The later phases of the response corresponded with peak accumulation of eosinophils, neutrophils, macrophages and myofibroblasts/hepatic stellate cells (HSCs) and the expression of chemokines with activity for these cells including CCL11, members of the Monocyte-chemoattractant protein family (CCL7, CCL8, CCL12) and the hepatic stellate cell/myofibroblast chemoattractant CXCL1. Peak expression of macrophage chemoattractants (CCL6, CXCL14) and markers of alternatively-activated macrophages (e.g. Retnla) during this later phase provided further evidence for a role for these cells in schistosome-induced pathology. Additionally, I demonstrated that CCL7 immunolocalises to the fibrotic zone of granulomas. Furthermore, striking up-regulation of neutrophil markers and the localisation of neutrophils and the neutrophil chemokine S100A8 to fibrotic areas suggested the involvement of neutrophils in S. japonicum¬-induced hepatic fibrosis. The transcriptional profile of the spleen was closely related to changes in cellular composition illustrated by flow cytometry and immunohistochemistry. Significant up-regulation of genes associated with progression through the cell cycle, proliferation makers and genes involved in lymphocyte proliferation, paralleled the initial expansion of T-cells and B-cells and the increased cellularity of the spleen overtime. Accumulation of eosinophils, neutrophils and macrophages was paralleled by enhanced expression of markers for these cells and the declining proportion of B- and T-cells in the spleen over time was reflected in the decreased expression of B- and T-cell markers. Significant up-regulation of Chi3l3 and F4/80+ macrophages suggested the presence of alternatively activated macrophages in the spleen, where these cells could play an immunoregulatory role. Comparison of the liver and spleen profiles revealed divergent expression of chemokines and cell adhesion molecules. Expression of lymphocyte chemokines including the homeostatic chemokines, CXCL13, CCL19 and CCL21, were significantly up-regulated in the liver while down regulated in the spleen. Expression of chemokines with activity for eosinophils (CCL11, CCL24), neutrophils (CXCL1) and monocytes (CXCL14, CCL12) and the cell adhesion molecules VCAM1, NCAM1, PECAM1 were up-regulated in the liver while unchanged in the spleen. Chemokines up-regulated in both organs were expressed at significantly higher levels in the liver. Divergent expression of chemokines and cell adhesion molecules likely contributes to the development of a chemotactic signalling gradient that promotes recruitment of effector cells to the liver. The results of liver and spleen microarrays suggested an important role for alternatively activated macrophages in the development of schistosome-induced pathology. This led me to investigate the in vivo transcriptional profile of S. japonicum SEA-stimulated peritoneal macrophages. The transcriptional profile of these cells was characterised by up-regulation of alternatively activated macrophage makers (Chi3l3, Chi3l4, Arg1). Retnla was not significantly induced in these macrophages suggesting that the specific function of these cells may differ to those induced by S. mansoni and other parasites. Other features of the transcriptional profile of these cells included modulated expression of T-cell co-stimulatory molecules and chemokines which may confer immunomodulatory activity. S. japonicum-stimulated alternative activation of macrophages was additionally associated with deactivation of classical activation pathways and altered expression of cell surface receptors and complement components that may alter phagocytic activity. Together these data significantly enhance our understanding of the mechanisms associated with alternative activation of macrophages and provide significant insight into the role of these cells in schistosomiasis japonica. The findings presented in this thesis represent the most comprehensive description to date of the molecular mechanisms, and especially chemotactic signalling pathways, regulating the development of schistosome-induced granulomas, fibrosis, splenomegaly and alternative macrophage activation in the murine host. In summary, my data have revealed that co-ordinated gene expression of chemokines in the liver and spleen regulates the recruitment of cells to the liver during schistosome infection. My results provide additional evidence for a role for neutrophils and alternatively activated macrophages in the development of schistosome-induced pathology and provide further insight to the molecular basis of alternative macrophage activation during infection. Furthermore, my data serve to highlight clear differences in the pathogenesis of schistosomiasis mansoni and schistosomiasis japonica. Together these findings further our understanding of the systemic, local, cellular, and especially, chemokine signalling pathways that regulate the development of S. japonicum-induced pathology and offer correlative insight into the pathogenesis of other chronic inflammatory diseases where fibrosis, splenomegaly and alternative activation of macrophages are common features.

Diss. (sammanfattning) Stockholm : Karol. inst., 2002. / Härtill 6 uppsatser.

Thesis (Ph. D.)--University of Washington, 2000. / Vita. Includes bibliographical references (leaves 111-119).

Made available in DSpace on 2014-06-11T19:30:30Z (GMT). No. of bitstreams: 0 Previous issue date: 2006-06-28Bitstream added on 2014-06-13T19:19:24Z : No. of bitstreams: 1 campos_cfm_dr_jabo.pdf: 893698 bytes, checksum: 797044f736c6cdcb2636bfe07a8e23cf (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Neste trabalho são apresentados dados de abundância média, prevalência e intensidade média de parasitismo em trinta e quatro espécimes de Pseudoplatystoma fasciatum, popularmente conhecidos como cacharas, capturados nos meses de março, setembro e novembro de 2003, outubro de 2004 e 2005, com tarrafas e redes de espera no rio Aquidauana. Todos os indivíduos amostrados encontravam-se parasitados. Dezenove espécies de parasitos foram identificadas, sendo sete cestodas proteocefalídeos, três monogeneas, um digenea, três nematodas, um acantocéfalo, um crustáceo, um pentastomídeo e duas espécies de mixosporídeos. As espécies de cestóides, Harriscolex kaparari e Megathylacus travassosi são registradas pela primeira vez para P. fasciatum capturados no rio Aquidauana. Os monogeneas foram os endohelmintos que apresentaram maior prevalência (100 %), seguidos pelos cestodas (91,18 %) e nematodas (58,82 %). / In this work are presented mean abundance, prevalence and mean intensity of infection in 34 specimens of Pseudoplatystoma fasciatum, commonly called cacharas. Fish were captured in Aquidauana river in March, September and November 2003, October 2004 and 2005, with nets in the Aquidauana river. All analyzed fish were infected. Nineteen species of parasites were collected, seven of proteocephalid cestodes, three of monogeneans, one of digeneans, three of nematodes, one of acanthocephalan, one of crustacean, one of pentastomid and two species of myxosporeans. The first record of Harriscolex kaparari and Megathylacus travassosi in P. fasciatum in the Aquidauana river was observed. Monogeneans were showed the highest prevalence, followed by cestodes (91.18 %) and nematodes (58.82 %).

Submitted by ISABELLA BARBOSA DOS SANTOS null (isabellabs853@hotmail.com) on 2018-04-04T23:51:02Z No. of bitstreams: 1 dissertação_Isabella_Barbosa_dos_Santos.pdf: 1903091 bytes, checksum: 01a14a6ac4969df8c86c454e56e542c4 (MD5) / Approved for entry into archive by Alexandra Maria Donadon Lusser Segali null (alexmar@fcav.unesp.br) on 2018-04-05T13:49:47Z (GMT) No. of bitstreams: 1 santos_ib_me_jabo.pdf: 1903091 bytes, checksum: 01a14a6ac4969df8c86c454e56e542c4 (MD5) / Made available in DSpace on 2018-04-05T13:49:47Z (GMT). No. of bitstreams: 1 santos_ib_me_jabo.pdf: 1903091 bytes, checksum: 01a14a6ac4969df8c86c454e56e542c4 (MD5) Previous issue date: 2018-02-22 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / A caprinocultura é considerada um segmento de grande importância em agronegócios no país, gerando importante fonte de carne e leite, principalmente na região Nordeste. Entretanto, este criatório sofre grandes perdas econômicas devido às parasitoses que acometem o trato digestório. Dentre estas, destaca-se, o gênero Haemonchus, cujas espécies H. contortus e H. placei parasitam abomaso de caprinos e bovinos, respectivamente, havendo possibilidade de infecção cruzada dessas duas espécies helmínticas. O objetivo do presente estudo foi avaliar a viabilidade do parasitismo por H. placei em caprinos e compará-la à patogenicidade por H. contortus. Foram utilizados 14 caprinos, recém-nascidos, mantidos em gaiolas metálicas de piso suspenso, assim distribuídos: GI - quatro cabritos inoculados com 5000 larvas infectantes (L3) de H. placei, GII – quatro infectados com 5000 larvas infectantes (L3) de H. contortus, GIII – quatro animais inoculados com 2500 larvas L3 de H. contortus + 2500 H. placei e GIV – dois cabritos que receberam apenas água (controle). Exames de contagem de ovos de estrongilídeos por grama de fezes (OPG) foram realizados diariamente, após o 7º dia da inoculação. Os períodos pré-patentes foram de 24 dias para H contortus e de 31 dias para H. placei. O grupo inoculado apenas com H. placei apresentou médias de OPG inferiores quando comparado aos demais grupos. Decorridos 42 dias pós-infecção, os 14 caprinos foram eutanasiados e necropsiados, sendo coletados “in totum” os exemplares de Haemonchus. O grupo I apresentou média de 25,5 espécimes H. placei, o grupo II média de 619,5 de exemplares de H. contortus, o grupo III média de 120 exemplares de H. placei e 604,75 de H. contortus e no grupo IV nenhum helminto foi diagnosticado. Não foram constatadas quaisquer outras espécies e nem formas imaturas de Haemonchus no trato gastrintestinais dos caprinos. Fragmentos do abomaso foram coletados e armazenados em formol tamponado para análise histopatológica. Foram observados no grupo I lesões microscópicas leves apenas com poucos focos inflamatórios. Nos grupos II e III foram diagnosticadas lesões extensas, como edemas, focos inflamatórios difusos, infiltrado de eosinófilos, diminuição das células parietais. No grupo IV nenhuma alteração histopatológica foi diagnosticada. Nas condições desse estudo, a espécie caprina foi razoavelmente susceptível ao parasitismo por Haemonchus placei, sendo esse parasitismo mais proeminente nos animais que receberam infecção mista (H. placei + H. contortus). / Goat breeding is considered a very important segment in Brazilian agribusiness, generating a significant source of meat and milk. However, this industry suffers great economic losses due to parasitic diseases that affect the digestive tract, compromising their development. Among these, the Haemonchus genus stands out, with H. contortus and H. placei parasitizing goats and cattle, respectively, with the possiblity of cross infection between these helminth species in abomasu of hosts. The objective of the present study was to estimate the viability of H. placei parasitism in goats, and compare it with H. contortus pathogenicity. Fourteen newborn goats were used, distributed in the following way: GI - four goats inoculated with infective larvae (L3) of H. placei; GII – four animals infected with infective larvae (L3) of H. contortus; GIII - four caprines inoculated with larvae L3 of H. contortus + H. placei; and GIV - two goats that received only water (control). Each animal received 5000 L3 of Haemonchus species, in a single dose, orally. Egg per Gram of Feces (EPG) exams were performed daily after the 7th day of inoculation, establishing that the pre-patent period of H. contortus was 24 days and H. placei was 31 days. The group inoculated only with H. placei presented lower EPG means when compared to the other groups. After 42 days post-infection, all 14 goats were euthanized and necropsied, and Haemonchus specimens were collected “in totum". Group I presented an average of 25.5 specimens, while the mean of group II was 619.5, group III obtained an average of 120 specimens of H. placei and 604.75 of H. contortus and group IV did not present any specimens. No other species and no immature forms of Haemonchus were found in gastrintestinal tracts of experimental animals. Fragments of abomasum were collected and stored in buffered formalin for histopathological analysis, in which GI group showed mild microscopic lesions with only a few inflammatory foci, while groups II and III had extensive lesions, such as edema, diffuse inflammatory foci, eosinophilic infiltrate and hyperplasia of parietal cells. Group IV showed no changes. Under conditions of this study, the caprine species did not present itself as susceptible to parasitism by Haemonchus placei, with such parasitism being more prominent when in consortium with Haemonchus contortus.

The Infectious Diseases of East African Livestock (IDEAL) project followed 548 East African Shorthorn Zebu (EASZ) calves in Western Kenya for the first year of life and monitored the sequelae of infections by multiple parasites. More than 50 different parasites were identified during this time. The IDEAL project also gathered environmental information about the farm and collected phenotypic data on the calf and its dam. Calves were also genotyped for 55,777 single nucleotide polymorphisms (SNPs). Recent research has looked at coinfection in rodents and humans but not in indigenous cattle. Here I investigate the evidence for coinfection in EASZ and study the associations occurring between coinfecting parasites. In addition, I examine the genetic and phenotypic factors which predispose an individual to infection with multiple parasites. Using information gathered by the IDEAL project, my thesis consists of the following chapters. An investigation of the nature of concurrent associations and of lagged effects between different parasites. Using the parasites Theileria spp., Coccidia spp., Strongyloides spp., strongyles and Calicophoron spp. I show that the patterns of association between different parasites are complex: there is evidence for both positive and negative associations. For example, infection with Strongyloides spp. increased the risk of strongyle infection. Conversely, in other cases, being infected with one parasite decreased the calf’s risk of infection with another parasite: for example, infection with Strongyloides spp. decreased the risk of infection with Calicophoron spp. A study of the relationship between different respiratory viruses and their effect upon the host. I confirm that positive associations exist between Infectious Bovine Rhinotracheitis (IBR), Bovine Viral Diarrhoea Virus (BVDV) and Bovine Parainfluenza Virus Type 3 (PIV3) in a previously unstudied setting; being seropositive for any one of these three viruses means that an individual is more likely to be seropositive for the other two viruses than expected by chance. Being seropositive for IBR, BVDV or PIV3 did not affect the average daily weight gain of the calf, nor did PIV3 and BVDV serostatus have an effect on the calf ever experiencing a clinical episode. However, IBR seropositive calves were less likely to experience a clinical episode of some form, suggestive of some protective aspect of IBR. An examination of the sources of variation in faecal strongyle egg counts (EPG), and their association with body weight, host genetics and a suite of haematological measures. Using estimates of relatedness derived from the SNP data, I established that strongyle EPG has a genetic basis in EASZ, with a heritability of 23.9% (S.E. = 11.8%) and showed a consistently strong negative association between strongyle infection and the haematological parameters white blood cell count, red blood cell count, total serum protein and absolute eosinophil count. Furthermore, calf body weight at 1 week old was a significant predictor of strongyle EPG at 16-51 weeks, with smaller calves being predisposed to a higher strongyle EPG later in life. A genome-wide association study (GWAS) to investigate if there is a genetic predisposition to East Coast Fever (ECF) death and a genetic basis to the packed cell volume at the time of seroconversion to Theileria parva (PCVTP). I found no robust evidence for a relationship between genotyped single nucleotide polymorphisms (SNPs) and ECF death or PCVTP. The effect of sample size upon GWAS and significance thresholds was investigated further through simulations. I conclude that the small number of cases influences the probability of association between a SNP and the phenotypic trait. Smaller case numbers produce more artifactual associations with SNPs, an effect not fully compensated for by the standard Bonferroni correction, suggesting that an empirical significance threshold should be used to directly account for sample size. The results of this thesis provide an understanding of the associations occurring between different parasites, and of their causes and consequences. I discuss the results in the context of their implications for disease control strategies, suggesting the benefits of an integrated approach to control worm and T. parva alongside the possible genetic selection for parasite resistance and supplementary feeding of lightweight individuals to improve the health of EASZ.

Oxyurid nematodes Aspiculuris tetraptera and Syphacia obvelata are both common mouse intestinal parasites; in the same location several species of trichomonads occur. Tritrichomonas muris is the most often found, but there are also some others: Tritrichomonas minuta, Pentatrichomonas hominis or Hexamastix muris. It is known that, under some circumstances, trichomonads can be found in the intestine of mouse pinworms, as reported by Theiler and Farber (1936) for T. muris in A. tetraptera and S. obvelata. What is interesting, trichomonads were found also in pinworms from trichomonad-negative mice. In 1933, Becker observed motile trophozoites of T. muris in the egg of S. obvelata. These observations offer a chance that the protist may be transmitted to a new host in similar manner (via pinworm eggs) as it was described for Histomonas meleagridis. In our study we document trichomonads in pinworm bodies, determine species of trichomonads, search for their localization, morphology and, based on the data, assess possibility of trichomonad transmission to a new mammalian host via pinworm eggs. By use of classical histology, in situ hybridization and transmission electron microscopy, we observed trichomonads exclusively in the intestinal lumen of both pinworm species. They were found in the entire pinworm...

Soil-transmitted helminth (STH) (A. lumbricoides, T. trichiura and hookworm) infections are a significant public health concern in Honduras. These infections are treatable using inexpensive anthelmintic medications, however long-term control and eradication will require large investments in public and private sanitation infrastructure. Importantly, both types of interventions are targeted towards high-risk populations and regions rather than individuals. The goal of this thesis is to contribute to improving the efficiency of soil-transmitted helminth control efforts in Honduras. In our first study, we use multiple regression analyses to identify determinants of STH infections and generate estimates of A. lumbricoides, T. trichiura and hookworm infection prevalence, as well as recommended deworming frequencies, for each of Honduras’ 298 municipalities. Our estimates suggest that prevalence of all three infections has declined over time, however 75% of municipalities still require annual or semi-annual deworming. In our second study, we quantify how the type of region used for measuring prevalence and allocating resources can impact the success and efficiency of public health programs. More specifically, we compare administrative regions to alternative zoning schemes at the same geographic scale. Our findings suggest that regions designed to be homogeneous with respect to prevalence can be more efficient than existing municipalities (at the same scale) for distributing resources. This research has implications on future control efforts. / Thesis / Master of Science (MSc)