Inclusion body hepatitis (IBH) has been occurring as an economically important, emerging disease of broiler chickens in several countries. Historically, IBH has been identified as a secondary disease, often associated with common immunosuppressive diseases. However, few studies have identified IBH as a primary disease with no apparent association with immunosuppressive diseases. The objectives of this study were to develop an animal model of IBH in commercial broilers, to demonstrate vertical transmission of fowl adenoviruses (FAdVs) in broiler breeders and to control IBH in broilers by vaccinating their parents with an inactivated FAdV vaccine. In order to develop an animal model of IBH in commercial broilers, fourteen-day old broilers were inoculated intramuscularly with 1x104 1x107 CCID50 of either FAdV x11a-like virus, two strains of FAdV-8a (FAdV-8a strain TR-59 and FAdV-8a strain T8-A) or FAdV-11strain 1047. Four days following FAdV inoculation, 5% - 15% mortality was observed and dead birds showed histologic lesions of hemorrhagic necrotizing hepatitis. This animal model reproduced the clinical disease, and pathological lesions of IBH that have been described in commercial broilers. In order to demonstrate vertical transmission of the FAdV, 35-week-old broiler breeders were inoculated with 1x106 CCID50 of either FAdV x11a-like virus, FAdV-8a strain TR-59, FAdV-8a strain T8-A or FAdV-11 strain 1047. Eggs from infected breeders were collected and hatched seven days post-inoculation. Clinical signs or mortality were not observed in parents; however broiler progeny derived from broiler breeders inoculated with FAdV-8a strain T8-A had 30% IBH mortality by seven days of age. The hexon gene loop 1 sequence of the virus isolated from affected broiler progeny showed 100% identity to FAdV-8a strain T8-A. In order to demonstrate protection of broilers against IBH by vaccination of their parents, four groups of broiler breeders were vaccinated with either FAdV-8a strain T8-A (2x107 or 2x104 CCID50) formulated with 20% oil-in-water emulsion, or FAdV x11a-like virus (2x107 or 2x104 CCID50) formulated with 20% oil-in-water emulsion at the age of 12 and 15 weeks. The control group was inoculated with 20% oil-in-water emulsion. Broiler progeny were challenged with FAdV-8a strain T8-A to study the immunoprotective effect of the vaccine. Although, survival of broilers following FAdV-8a strain T8-A challenge was not significantly different among vaccinated and non-vaccinated groups (P>0.05), immunoprotective effect was enhanced by the increase dose of FAdV antigens (P>0.05). Further studies are necessary to improve the vaccine efficacy to protect broilers against IBH.<p> In conclusion, the results of this study support the hypothesis that IBH in broilers in Canada is a vertically-transmitted primary disease with no known immunosuppressive involvement. The results also demonstrated that inactivated antigens of FAdV are able to partially protect broilers against IBH by vaccinating their parents. Further studies with different formulations, and priming the immune system of broiler breeders with live FAdV prior to vaccination with inactivated FAdV vaccines are necessary to improve the efficacy of inactivated IBH vaccine.

Inclusion body hepatitis (IBH) has been occurring as an economically important, emerging disease of broiler chickens in several countries. Historically, IBH has been identified as a secondary disease, often associated with common immunosuppressive diseases. However, few studies have identified IBH as a primary disease with no apparent association with immunosuppressive diseases. The objectives of this study were to develop an animal model of IBH in commercial broilers, to demonstrate vertical transmission of fowl adenoviruses (FAdVs) in broiler breeders and to control IBH in broilers by vaccinating their parents with an inactivated FAdV vaccine. In order to develop an animal model of IBH in commercial broilers, fourteen-day old broilers were inoculated intramuscularly with 1x104 1x107 CCID50 of either FAdV x11a-like virus, two strains of FAdV-8a (FAdV-8a strain TR-59 and FAdV-8a strain T8-A) or FAdV-11strain 1047. Four days following FAdV inoculation, 5% - 15% mortality was observed and dead birds showed histologic lesions of hemorrhagic necrotizing hepatitis. This animal model reproduced the clinical disease, and pathological lesions of IBH that have been described in commercial broilers. In order to demonstrate vertical transmission of the FAdV, 35-week-old broiler breeders were inoculated with 1x106 CCID50 of either FAdV x11a-like virus, FAdV-8a strain TR-59, FAdV-8a strain T8-A or FAdV-11 strain 1047. Eggs from infected breeders were collected and hatched seven days post-inoculation. Clinical signs or mortality were not observed in parents; however broiler progeny derived from broiler breeders inoculated with FAdV-8a strain T8-A had 30% IBH mortality by seven days of age. The hexon gene loop 1 sequence of the virus isolated from affected broiler progeny showed 100% identity to FAdV-8a strain T8-A. In order to demonstrate protection of broilers against IBH by vaccination of their parents, four groups of broiler breeders were vaccinated with either FAdV-8a strain T8-A (2x107 or 2x104 CCID50) formulated with 20% oil-in-water emulsion, or FAdV x11a-like virus (2x107 or 2x104 CCID50) formulated with 20% oil-in-water emulsion at the age of 12 and 15 weeks. The control group was inoculated with 20% oil-in-water emulsion. Broiler progeny were challenged with FAdV-8a strain T8-A to study the immunoprotective effect of the vaccine. Although, survival of broilers following FAdV-8a strain T8-A challenge was not significantly different among vaccinated and non-vaccinated groups (P>0.05), immunoprotective effect was enhanced by the increase dose of FAdV antigens (P>0.05). Further studies are necessary to improve the vaccine efficacy to protect broilers against IBH.<p> In conclusion, the results of this study support the hypothesis that IBH in broilers in Canada is a vertically-transmitted primary disease with no known immunosuppressive involvement. The results also demonstrated that inactivated antigens of FAdV are able to partially protect broilers against IBH by vaccinating their parents. Further studies with different formulations, and priming the immune system of broiler breeders with live FAdV prior to vaccination with inactivated FAdV vaccines are necessary to improve the efficacy of inactivated IBH vaccine.

No description available.

Sporadic Inclusion Body Myositis (sIBM) is an inflammatory muscle disease that strikes individuals at random and accounts for approximately 1/3 of all idiopathic inflammatory myopathies. It is characterized by progressive weakness of distal and proximal muscles and is the most common muscle disorder in individuals over 50 years of age. Currently, there is no known cause, cure, or enduring treatment for sIBM, although a number of theories as to its cause have been proposed. One theory proposes that activation of the inflammatory/ immune response is the primary trigger resulting in muscle degeneration and protein abnormalities, while an alternative theory suggests that sIBM is a degenerative muscle disease with abnormal pathogenic protein accumulation, in particular Abeta, being a primary cause that triggers an inflammatory/ immune response. Mitochondrial abnormalities have been observed in skeletal muscle from patients diagnosed with the disease, however the role of the mitochondria in disease pathology is still unclear. The aim of this dissertation was to evaluate: 1) the role of the mitochondria in the development of sIBM and 2) the role of amyloid beta on mitochondrial function in skeletal muscle. A better understanding of the role of the mitochondria in the development of sIBM may help to identify novel prevention and/ or treatment strategies. / Ph. D.

Diss. (sammanfattning) Stockholm : Karol inst., 2003. / Härtill 4 uppsatser.

[Truncated abstract] Sporadic inclusion body myositis (sIBM) is a chronic inflammatory disease that is the most common myopathy in individuals above the age of 50 in the Caucasian population. sIBM is characterised by cytotoxic immune infiltration of skeletal muscle, consisting primarily of CD8+ T-cells and macrophages, as well as a degenerative process, with muscle fibre vacuolation and intracellular filamentous inclusions. The pathogenesis of sIBM is likely to involve a complex interaction between genetic and environmental factors. Whilst the physiological and pathological characteristics of sIBM have been clearly identified, the exact origin and genetic basis of the disease remains unknown. A number of studies show that sIBM is associated with alleles of the major histocompatibility complex (MHC) on chromosome 6p21.3 and specifically with two ancestral haplotypes (AH) in Caucasians – the 8.1AH, defined by HLA-B*0801, HLA-DRB1*0301 and the 35.2AH, defined by HLA-B*3501, HLA-DRB1*0101. Mapping studies subsequently showed that sIBM susceptibility likely originates from a 389kb region of the MHC, spanning from centromeric of PBX2 to telomeric of HLA-DRB1. The central hypothesis of this thesis was that susceptibility to sIBM is conferred by a single allele found within a region defined using the 8.1AH, which is also carried by other haplotypes associated with sIBM. Three patient cohorts from Australia, the U.S.A and Japan were studied. ... Of the 32 alleles genotyped, none were found in all susceptibility haplotypes and one was common, but not unique, to the 8.1AH, 7.2AH and 52.1AH. Five SNPs were also found in two of the three haplotypes, although none were specific to the sIBM susceptibility haplotypes. These data suggest that the 8.1AH is likely to carry an sIBM susceptibility allele independent of the 35.2AH, 7.2AH and 52.1AH. Based on the possible mechanism of action in cellular differentiation and its location within the 8.1AH-defined sIBM susceptibility region reported in 2004, NOTCH4 was a strong candidate for conferring sIBM susceptibility. NOTCH4 coding region polymorphisms were thus investigated in a Caucasian patient cohort to assess any possible role in sIBM susceptibility. While the frequency of some alleles were increased in sIBM patients, the strong linkage disequilibrium throughout the MHC prevented confirmation of any alleles as playing a direct role in sIBM. The 8.1AH-derived sIBM susceptibility region was further refined using recombination mapping. This approach used markers characterised against multiple haplotypes to genotype patients carrying part of the 8.1AH to locate a common, overlapping susceptibility region. Recombination mapping of patients revealed a common overlapping region of the 8.1AH, extending from BTNL2 to HLA-DRB3. The results of the study indicate that 8.1AH-derived susceptibility for sIBM is likely to originate from a 172kb region encompassing HLA-DRA, HLA-DRB3 and part of BTNL2. These genes warrant further investigation in future studies.

Sporadic inclusion body myositis (IBM) is the commonest acquired myopathy in individuals aged over 50 years. Currently, diagnosis is based largely upon specific muscle biopsy findings, though these are thought to lack sensitivity. More recently, many abnormal protein aggregates have been described in IBM and some have been proposed as diagnostic markers. However, their diagnostic utility is untested and how they relate to the pathogenesis is uncertain. The clinical characteristics, pathological findings and disease course in 67 patients with histopathologically diagnosed and clinically diagnosed IBM were compared and revealed that patients with IBM have a characteristic pattern of weakness at presentation that is independent of the pathological findings. Current diagnostic pathological features are more common in older patients and therefore may be linked to disease duration. The pathology of IBM and the diagnostic utility of protein aggregates in IBM were studied in six clinicopathologically typical cases of IBM. Protein aggregates immunoreactive for p62, TDP-43, myotilin, α B-crystallin and ubiquitin were more abundant than the current diagnostic pathological features. The diagnostic potential of these pathological features was then assessed in pathologically typical and atypical IBM and controls. This revealed that staining for p62, MHC Class I, CD8 positive lymphocytes and mitochondrial abnormalities could support the diagnosis and should be included in diagnostic criteria for IBM. Laser microdissection and label-free mass spectrometry were used to validate the histological findings. The presence of nuclear and aggresomal proteins in rimmed vacuoles indicated that they are derived from myonuclei. Increased protein synthesis and ER stress suggest that abnormal protein aggregates may be the result of abnormal protein production. In conclusion, this thesis emphasises that a diagnosis of IBM can be confidently made on clinical grounds, with a supportive muscle biopsy. The histological and proteomic findings reveal that there is a spectrum of pathological features most likely associated with the disease duration and that adherence to the current pathological based diagnostic criteria may have led to some erroneous conclusions about the pathogenesis of this enigmatic disease.

Expression of foreign proteins in host organisms usually results in the development of insoluble, inactive proteins. Further, these proteins have a tendency to form aggregates termed inclusion bodies. However, the formation of inclusion bodies can be avoided by fusing the gene encoding the foreign protein to a highly soluble protein. In this report Sterol Carrier Protein-2 (SCP-2) is reviewed as a possible solubility tag. The experiment was carried out by fusing SCP-2 to one of two i nsoluble proteins, Green fluorescent protein (GFP) or a form of chloramphenicol acetyl transferase (CAT∆9). The protein fusion was then inserted into the vector pET-15b, transformed in Escherichia coli and the yield of actively expressed protein was measured. The results obtained from this study, as evaluated by PageBlue staining and  Western blot, are indicating that SCP-2 does not improve the solubility of GFP or CAT∆9. Nonetheless, the solubility of GFP has earlier been increased by fusing it to the solubility tag maltose-binding protein (MBP).  Producing more soluble forms of CAT∆9  have also been tested but without success. Therefore the conclusion drawn from this experiment is that SCP-2 does not work as a solubility tag, however more research must be performed to conclude this with certainty.

Elastin-like peptide (ELP) fusions promote therapeutic delivery and efficacy. Recombinant proteins, like neurotrophins, lack bioavailability, have short in vivo half-lives, and require high manufacturing costs. Fusing recombinant proteins with genetically encodable ELPs will increase bioavailability, enhance in vivo solubilization, as well as provide a cost-effective method for purification without the need for chromatography. During expression of neurotrophin-ELP (N-ELP) fusions, dense water-insoluble aggregates known as inclusion bodies (IBs) are formed. Inclusion bodies are partially and misfolded proteins that usually require denaturants like Urea for solubilization. Strong denaturants arrest ELPs stimuli-responsive property and increase unwanted aggregation, making purification difficult, yet possible. The current field of study exhibit issues with protein recovery due to solubility issues and aggregation. This study examines the solubility challenges of inclusion body proteins and the role ELP fusion tags play on IBs solubility. Elastin-like peptides are a class of stimuli-responsive biopolymers whose biocompatibility and limited toxicity are attractive for biological applications. ELPs are tunable polymers, which consist of peptide repeat units (VPGXG), where X is any amino acid except Proline while the guest residue or length of the sequence can be chosen. ELPs have uniquely tunable phase transitioning properties that allow the protein to undergo molecular self-assemblies into different nanostructures in response to the changes in their environment (e.g. pH or temperature). Optimizing the purification process via suppressing aggregation during the refolding process has increased protein recovery slightly however, more work is needed to attain 90 percent recovery. Usage of ELPs has increased the solubility of N-ELP fusions, specifically for brain-derived neurotrophic factor ELP fusions.

No description available.