Molecular characterization of porcine genes encoding complement components of the terminal lytic pathway and their association with hemolytic complement activity

Activation of the complement system from three different pathways (classical, alternative and lectin pathway) results in the generation of the C3-convertase enzyme, which plays a key role in formation of the membrane attack complex (C5b-C9) causing the death of target cells. The porcine C3 and C5 complement components were characterized and studied for association with hemolytic complement activity (Kumar et al. 2004, Wimmers et al. 2003). In order to gain understanding for the membrane attack complex action in the innate immune mechanism, in this study it was focussed on the terminal complement components C6, C7, C8, and C9 to characterize their molecular structure, to detect single nucleotide polymorphisms (SNPs), to establish their location on chromosome, and to associate their genetic variation with hemolytic complement activity in both classical and alternative pathway in the pig. The entire length of cDNA sequence of the candidate genes C6, C7, C8A, C8B, C8G and C9 were identified with 3306, 3561, 2146, 2461, 840 and 2536 bp encoding 935, 843, 589, 611, 202 and 543 amino acids, respectively. The porcine deduced protein sequence of the candidate genes showed 67-83% identities with human analogue. Respectively, screening the coding region revealed five, six, seven, nine, and two SNPs in the porcine C6, C7, C8A, C8B, and C9 but non in C8G by polymerase chain reactionrestriction fragment length polymorphism (PCR-RFLP). Most of the SNPs belong to the functional protein domains such as TSP1, LDLa, MACPF, CCP and FIMAC. Genotyping for several SNP sites in three porcine breeds German Landrace (LR), Pietrain (PIE) and Muong Khuong (MK) showed that European breeds (LR and PIE) had higher allelic variation than the Asian breed (MK). All genotypic frequencies fit to Hardy-Weinberg equilibrium rule. Using the INRA-Minnesota porcine Radiation Hybrid mapping panel, the porcine C6, C7, and C9 were assigned to the q-arm of chromosome 16 (q1.4) whereas the porcine C8A, and C8B were mapped to chromosome 6 (q3.1-q3.5). Particularly the porcine C8G was located on chromosome 1 (q2.13). Genetic association with hemolytic complement activity in both classical (CH50) and alternative pathway (AH50) was carried out in 417 animals of a F2 DUMI resource population derived from cross between Duroc and Berlin Miniature Pig. Therefore, the F2 DUMI animals were immunized with Mycoplasma hyopneumoniae (Mh), Aujeszky (ADV) and porcine reproductive and respiratory syndrome (PRRSV) vaccine. Sera were isolated from blood samples taken prior and post vaccinations and measurement for CH50 and AH50 was conducted thereafter. For each gene except the porcine C8G, the SNP site with amino acid substitution 862A→G for C6, 881A→G for C7, 1544C→T for C8A, 222C→T for C8B, and 407C→G for C9, segregating in the DUMI, were used for genotyping the F2 animals using PCR-RFLP with the restriction enzymes TaqI, MboII, Hin6I, FnuDII, and HpyCH4III, respectively. The association results illustrated that significant difference in hemolysis among genotypes was found in CH50 for C7 (p=0.0080), and C9 (p=0.0488). However, this was close to significance for C6 (p=0.0853) and C8A (p=0.0650) in CH50. Therefore between homozygous genotypes CC and TT for C8A hemolytic activity showed significant difference (p=0.0522). There was no association of any of the candidate gene with hemolytic complement activity in the alternative pathway. Analyzing the interaction between genotypes and eight different immunization time points in AH50 revealed significant differences for C8A (p=0.0027), C8B (p=0.0231), and C9 (p=0.0340) whereas in CH50 this interaction was found significant for C8B (p=0.0048). Hemolytic complement activity showed the highest values at the fourth day after immunization with ADV vaccine for CH50 whereas linear increment during the experiment was performed for AH50. Along the vaccination program after each of complement stimulation by different vaccines, a short termed increment of complement activity was found, especially with ADV vaccine. Also male animals always performed higher hemolysis than females in both pathways. These results show that hemolytic complement activity depends on the genetic variation, sex, age, kind of vaccine, and interaction of complement components. In summary, the obtained results provide the means for further understanding the role of C6, C7, C8, and C9 in natural immune response of the host against pathogens. It also promotes the porcine C6, C7, C8, and C9 as candidate genes in efforts to genetically improve general animal health, a goal of breeding programmes for food animals

Exploring Epigenetics as a Tool for Population Assessment and Conservation in Large Marine Predators

Worldwide, many large marine predator populations are in decline. These populations can be difficult to study due to the extensive home ranges and migration patterns often exhibited by these species. Molecular tools are therefore necessary to measure specific parameters on these populations that would otherwise be nearly impossible to obtain. This dissertation pioneers the use of environmental epigenetic approaches for that purpose, and specifically the epigenetic modification known as DNA methylation, using sharks and small cetaceans as model organisms. This work is organized into five chapters. Chapter I is an introductory chapter that lays out the fundamentals of environmental epigenetics and the future directions of the field. Chapter II is a study that culminated in the Bottlenose epigenetic aging tool (BEAT) which allows for the estimation of age in bottlenose dolphins. Chapter III extends epigenetic aging research to a non-model organism, Lemon sharks (Negaprion brevirostris), using a global DNA methylation quantification technique (MSAP) to assess age group differences in DNA methylation. This work provides the first evidence that the role epigenetics plays in aging for mammals, birds, and fish may also exists for sharks. Chapter IV is a study that explores the DNA methylation response in juvenile Lemon sharks to a major dredging event that occurred in Bimini, Bahamas. This study paves the way for incorporating epigenetics into ecological studies for long term monitoring of environmental changes. Chapter V summarizes the dissertation, showcasing how work has continued from previous chapters, and discusses the next direction of the work into the future

Association of the Major Histocompatibility Complex with Autism

The pathogenesis of autism has proven difficult to characterize. However, in many recent studies, it is suggested that the onset of this disorder is the result of multiple etiological factors, which include genetic, immunologic, and viral elements. Possible immunological influences found in subpopulations of patients with autism include decreased lymphocyte responsiveness, reduced natural killer cell activity, abnormal response to rubella vaccine, abnormal immune response to brain tissue, and decreased plasma levels of the fourth component of complement(C4). These aberrations and others imply a possible autoimmune mechanism in some cases for the development of autism. C4 deficiencies have been found in subjects with established autoimmune disorders, such as systemic lupus erythematosus and chronic active hepatitis, in recent investigations. There is also evidence that the major histocompatibility genes play an intimate role in autoimmune processes. Therefore, in knowing that the C4 genes are closely linked to the major histocompatibility genes, this study determined and analyzed the human leukocyte antigen profile of autistic patients, their siblings, and parents. In this study, it was found that the C4B complement null allele occurred in autistic patients at nearly twice the frequency compared to normals. However, the C4A complement null allele frequency was not found to be significantly altered. Several extended haplotypes were represented within the patients studied. However, the extended haplotype B44- SC30-DR4 was the only one found at a significantly increased frequency. Further investigations are needed to better understand the significance of these findings

Genetic signatures in a perennial ryegrass (Lolium perenne) population following recurrent selection for compatibility with an endophyte (Epichloë spp.) from tall fescue : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Plant Breeding at Massey University, Palmerston North, New Zealand

Table 1.1 and Figure 2.1 are re-used with permission.Perennial ryegrass or Lolium perenne L. (Poaceae) is the most extensively grown forage especially in the temperate regions of the world, including New Zealand. The development of forage cultivars is important to New Zealand since the livestock industry depends on perennial ryegrass for its nutrition needs. Among forage breeding objectives, persistence is particularly complex. It refers to the stability of dry matter yield over time. It is economically important because reseeding and cultivation can be capital-intensive. Persistence is partly modulated by the interaction of perennial ryegrass with Epichloë spp. as these fungal endophytes confer insect resistance for a more stable yield. Genetic factors in the host influence fungal biomass, alkaloid concentration, and endophyte vertical transmission frequency. The symbiotic relationship is therefore exploited in perennial ryegrass breeding. Thus, the objective of this study is to investigate a perennial ryegrass breeding population under recurrent selection (RS) for compatibility with an endophyte sourced from tall fescue. Specifically, this study aims to (1) investigate the transmission of the Epichloë sp. FaTG-3 strain AR501 in the breeding population PGG04, and (2) to examine how genetic variation changes during RS in terms of population differentiation. Since the selection program targets endophyte compatibility, signatures of selection that may be associated with the grass-endophyte interaction were also determined. It was hypothesized that: there will be a reduction of diversity, and an excess of rare alleles. Furthermore, it was hypothesized that loci under positive selection will have higher fixation index (FST), and their genotypes will be more correlated with the components of PCA- based population structure analysis compared to neutral loci. The presence of AR501 was examined in seeds, in the growing tillers, and by microsatellite genotyping for both the early and late generations of PGG04. The seed squash assay revealed that more than 90% of PGG04 seeds harboured the endophyte, regardless of the generation. Viable endophyte detection using tissue-print immunoblotting showed an increase in infection from ca. 5% to 33% between the early and late generations. Thus, the results suggest that positive selection for endophyte compatibility increased the proportion of viable endophyte in the population. This study provides evidence supporting host genetic control of the association in grass-endophyte interaction, and that this can be exploited in plant breeding programs. Changes in the genetic variation of PGG04 was investigated by comparing GBS data of the early and late generations. Results showed that selection enriched the late generation with rare alleles (0.02 - 0.08) compared with the early generation. Also, selection reduced expected heterozygosity from 0.3069 in PGG04-C2 to 0.3033 in PGG04-C6. Further, selection changed the population structure based on UPGMA dendrogram, PCA, and the model-based clustering method implemented in STRUCTURE. A few single nucleotide polymorphisms (SNPs) have relatively larger contribution to the population structure changes hence, they have relatively high FST, and their genotypes correlated with principal components. Logistic regression of these SNPs with infection data identified nine SNPs to be associated with the trait. Depending on the allele frequency, these SNPs can increase the odds of favourable infection by more than five times. Annotation of these SNPs identified S7_160751877 to be tagging an ABCG transporter gene. Since some ABC transporters mediate plant-microbe interactions, it is possible that the identified SNPs are tagging a gene involved in the host genetic control of grass-endophyte interaction

The chemoprophylaxis of meningococcal disease in the Cape Town City Council area : an evaluation of programme efficacy

This dissertation reports the findings of a study which was carried out in the Cape Town City Council area, in order to establish whether the offering of rifampicin to household contacts, of patients with meningococcal disease, resulted in protection of those contacts against developing the disease during a 32 week follow up period. The study took the form of a retrospective follow up of 3 350 household contacts of 412 cases notified over a 4-year period (mid 1988-mid 1992). It was found that the offering of rifampicin to the household contacts resulted in an odds ratio of not developing meningococcal disease over the 32-week follow up period of 14, 17 (SD = 12, 34). Although there was a tendency for contacts who were not offered rifampicin to have been younger, and of male gender, when compared to those who were offered prophylaxis, these demographic differences were not statistically significant at the 0,05 level. Furthermore, three out of the four male second cases, all in the younger age group, were in fact offered prophylaxis. It seems desirable that prophylaxis should be given as soon as possible. It is concluded, therefore, that the offering of rifampicin to household contacts of patients with meningococcal disease, living under the prevailing social circumstances in the Western Cape, has protective benefit for those contacts. It is likely that the chemoprophylaxis programme prevented up to 88 cases of meningococcal disease over the study period of four years, as well as preventing 8 deaths from this disease, in the CCC population

Characterization of mouse major urinary protein genes

Major urinary protein (MUP) genes were isolated from C57 genomic libraries, characterized by restriction enzyme mapping and compared with MUP genes isolated from BALB/c genomic libraries (Clark et al, 1982; Bishop et al, 1982). The conclusions drawn from the characterization of this new set of MUP genes are in agreement with those previously drawn from studies on the BALB/c MUP genes.Most MUP genes were found to share extensive homology in their transcription units and 5' and 3' flanking regions. Exceptions were those genes whose coding regions have been interupted by insertions and/or deletions. The MUP genes fall into two main groups based on hybridization criteria: group 1 and group 2 (Bishop et al, 1982). With the exception of one group 2 gene (BL-25/CL-2), restriction site homology was found to be greater within groups than between than. Restriction site homologies further divided the group 1 genes into two sub-groups. Sequence data revealed that the two sub-groups have different forms of an A-rich region located M0bp upstream of the TATA box.Messenger RNA from tissues that express MUP was shown to be more homologous to group 1 coding sequences than to group 2 coding sequences. In the liver, two forms of MUP mRNA can be distinguished. Group 1 sequences hybridized preferentially to the abundantly transcribed long form of the mRNA, while group 2 sequences hybridized preferentially to the short and rarer form of the mRNA. Genomic digests illustrated that two types of variation are found between the MUP genes of BALB/c and C57BL/Fa mice. The first relates to the presence of variant restriction fragments. Two cloned MUP genes carrying such fragments were identified. The second relates to variation in the intensity of common restriction fragments. Differences between the strains in the total number of MUP genes were not observed. Variation in the intensity of common restriction fragments are proposed to be the result of different homogenization events that took place in the mouse lineages from which BALB/c and C57BL/Fa were derived

A STUDY OF THE ROLE OF CYTOKINES IN ACUTE PANCREATITIS IN MAN

Introduction: Acute pancreatitis is an inflammatory disease with a diverse aetiology and variable clinical course. The IL-l gene cluster has been implicated in this disease. Aims: The aims of the study were to investigate polymorphisms of the genes encoded within the IL-l gene cluster in patients with acute pancreatitis and normal controls and to determine the relationship between the polymorphisms and protein levels. Methods: Genotype and allele frequencies were determined in controls (n=217) and patients with acute pancreatitis (n=137) using the polymerase chain reaction (PCR) followed by digestion with restriction endonucleases where applicable. Protein levels were determined using in vitro stimulation of PBMCs followed by Enzyme Linked Immunosorbent Assay (ELISA). Patients were categorised according to severity, organ failure scores and aetiology. Results: Allele l of the VNTR86 polymorphism in the IL-l RN gene was significantly increased in the severe group of patients compared to controls (81.9% vs 63.0%, x2=9.38, p=0.002, Pc=0.004) and in the idiopathic group compared to controls (82.4% vs 63.0%, x2=9.33, p=0.002, Pc=0.004). The polymorphisms within the genes and between the genes were strongly linked. Significantly more of the Mspl-VLP-VNTR86-Sspl 2-3-2-2 haplotype was observed in the control (15.7% vs 0.1%, x2 =2528.11, p<0.000000l, Pc<0.0000001) and patient (14.0% VS 0.1%, x2 =4368.10, p<0.000000l, Pc<0.0000004) populations than expected. Significantly more of the Pstl-Aval-Alul-Taql 2-2-2-1 haplotype was observed in controls (27.7% vs 9.7%, x2 =31.39, p<0.000000l, Pc<0.0000005) and patients (12.5% vs 2.0%, x2=53.69, p<0.000000l, Pc=0.0000007) than expected. Preferential combinations of the genotypes existed within controls and patients. The median IL-lα and IL-lβ protein levels from unstimulated PBMCs were significantly increased in patients compared to controls: median values (interquartile range). In the IL-lα study, significant differences were found at 24 hours: 193.5 (127.5-363.5) pg/ml vs 1.0 (0.0-3.0) pg/ml, p=0.005, 48 hours: 256.5 (171.5-417.0) pg/ml vs 6.5 (2.0-16.0) pg/ml, p=0.006 and at 72 hours: 210.5 (138-427) pg/ml vs 0.5 (0-7) pg/ml, p=0.005. In the IL-l β study, significant differences were found at 24 hours: 663 (507-782) pg/ml vs 12 (5-53) pg/ml, p=0.004, 48 hours: 620 (570-1080) pg/ml vs 14.5 (11-36) pg/ml, p=0.004 and at 72 hours: 545.5 (442-771) pg/ml vs 12.5 (2-43) pg/ml, p=0.006. Conclusion: Polymorphisms of the IL-l gene cluster are associated with susceptibility to and/or severity of the acute pancreatitis. Polymorphisms within the IL-l gene cluster are in linkage disequilibrium. Unstimulated PBMCs from patients with acute pancreatitis secrete significantly more IL-la and IL-IP protein levels compared to those from controls. The (AC)n, Alu I and VNTR86 polymorphisms do not correspond to differences in functional protein levels