Ypač svarbią reikšmę Lietuvos žemės ūkiui turi viena iš gyvulininkystės šakų – galvijininkystė. Jos plėtrai didelę žalą daro galvijų užkrečiamosios, ypač virusinės, ligos, todėl būtina greitai, tiksliai ir efektyviai diagnozuoti galvijų virusines ligas, užkirsti kelią jų plitimui Lietuvoje. Kvėpavimo ir virškinimo trakto ligomis galvijų prieauglis serga visose pramoninės gyvulininkystės šalyse, dėl šių ligų galvijai gaišta žymiai dažniau, nei nuo reprodukcijos, medžiagų apykaitos ligų ar mastitų. Didesnį veršelių susirgimų skaičių ir gaišimą nuo enteritų bei respiratorinių ligų galima būtų paaiškinti dideliu virusinių ligų – GVD, IGR, PG-3, RSV, adenovirusų ,RV, CV ir bakterinių infekcijų išplitimu. Mūsų darbo tikslas buvo atlikti galvijų užkrečiamųjų virusinių ligų paplitimo analizę, ištirti CV, RV, RSV ir adenovirusų paplitimą galvijų bandose. Dėl rota ir korona virusų buvo ištirti 56 išmatų mėginiai, dėl antikūnų prieš adenovirusą – 20, RS virusą 28 kraujo serumo mėginiai. Šiems tyrimams buvo naudojami komerciniai standartizuoti imunofermentinės analizės (IFA) rinkiniai. Veršelių 30 kraujo serumo mėginių buvo ištirti pusiau kiekybiniu natrio sulfito precipitacijos metodu, dėl imunoglobulinų kiekio nustatymo. Tyrimai atlikti LSMU VA Užkrečiamųjų ligų katedroje ir Nacionalinio maisto ir veterinarijos rizikos vertinimo instituto Virusologijos skyriuje 2013 metais. Įvertinus padėtį 3 ūkiuose, kur buvo atlikti ligų tyrimai, nustatyta, kad rotavirusine infekcija vidutiniškai... [toliau žr. visą tekstą] / Cattle take a very important place in Lithuanian agriculture. Since the development of livestock can be seriously affected by contagious diseases, especially viral ones, it is highly important quickly, accurately and efficiently diagnose viral disease of cattle, and to prevent its spreading in Lithuania. In all livestock farming industry countries, cattle offspring are suffering from respiratory and digestive tract diseases. They are dying from these diseases more often than from reproductive, metabolic diseases and mastitis. Increasing calves’ cases and mortality rate from enteritis and respiratory diseases can be explained by the high viral diseases such as GVD, IGR, PG-3, RSV, adenovirus, RV, CV and bacterial infections spread. The goal of our study was to analyze the spread of livestock infectious viral diseases and examine the CV, RV, RVS and adenovirus prevalence in the cattle herds. 56 fecal samples were tested for the rotavirus and coronavirus, 20 – for antibodies against adenovirus, and 28 blood serum samples for RS virus. To test these samples, we used standardized commercial enzyme-linked immunosorbent assay (ELISA) kits. 30 blood serum samples of calves were analyzed by quantitative sodium sulfite precipitation method for the determination of immunoglobulin. The tests took place at LSMU VA Department of Infectious Diseases and National Food and Veterinary Risk Assessment Institute, Department of Virology in 2013. Cattle from 3 farms were assessed for anticipated... [to full text]

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Dendritic cells (DC) are potent antigen presenting cells involved in the initiation of the alloimmune response and organ transplant rejection. This thesis, has investigated pharmacological and genetic approaches to manipulate DC in order to generate tolerogenic DC which elicit poor allostimulatory activity as potential cell therapy agents to treat allograft rejection. In the first aspect of this study, human monocyte-derived DC were used to study the influence of Rapamycin (RAPA) on DC phenotype and function. This study showed that RAPA when added to monocytes prior to DC differentiation or after DC maturation generated tolerogenic DC as evidenced by the ability of these cells to induce T cell hyporesponsiveness. However, T cell hyporesponsiveness was associated with downregulation of costimulatory molecules only when added prior to differentiation and surprisingly was not influenced by the induction of CD4 ⁺FoxP3 ⁺ T cells. To assess the effects of RAPA on DC function in the transplant setting an in vivo chimeric model of ovine vascularised skin allograft transplantation was established in immunocompromised NOD/SCID mice as a host. This model was established as a preliminary model to acquire in vivo data prior to testing the effect of pharmacologically modified DC in the preclinical ovine model of renal allograft transplantation, also established in the host laboratory. Firstly, comparison of ovine DC obtained from cannulation of the prefemoral lymphatic vessels in sheep demonstrated that RAPA-modified ovine DC acted as poor stimulators of allogeneic ovine T cells similar to human DC treated with RAPA. Secondly, in NOD/SCID mice engrafted with ovine skin, the infusion of allogeneic ovine T cells together with RAPA-modified ovine DC reduced histological rejection in comparison to control DC. In the second aspect of this study, the effects of genetic manipulation of DC were investigated. In order to investigate the effects of genetic modification of DC, two isoforms of the human HLA-G molecule, HLA-G1 (membrane bound) and HLA-G5 (soluble isoform) were used to generate adenoviral vectors. Unexpectedly, both HLA-G isoforms expressed by human DC transfectants were unable to induce allogeneic T cell hyporesponsiveness in the mixed lymphocyte reaction (MLR). Surprisingly, in the MLR the allogeneic T cells acquired HLA-G1, but not HLA-G5, indicating that direct cell contact and membrane transfer from DC to T cells occurred (Trogocytosis). In addition to HLA-G1, costimulatory molecules (CD40, CD80, CD86 and MHC Class II) were also cotransferred from DC to allogeneic T cells. Accordingly, in secondary proliferation assays T cells immunoselected after co-culture with allogeneic untransfected DC (TUT) demonstrated potent antigen presenting activity when used as stimulators of autologous T cells (analogous to the indirect pathway of antigen presentation). In contrast to TUT, immunoselected T cells that acquired HLA-G1 (THLA-G1) upon co-culture with DCtransfectants showed poor stimulatory capacity. Thus the data reported in this thesis supports the proposed novel concept that HLA-G acquired by T cells through genetically modified DC, functions to autoregulate T cells via T-T cell interaction through the HLA-G receptor ILT2 (negative signalling receptor) expressed on T cells. In conclusion, this thesis has firstly provided supportive evidence that the pharmacological modification of human and ovine DC with RAPA has potential therapeutic effects on allograft rejection. Secondly, the genetic modification of DC to induce expression of HLA-G has specifically allowed the transfer of this molecule to T cells by trogocytosis and the inhibition of alloreactive T cell expansion. / Thesis (Ph.D.) -- University of Adelaide, School of Medicine, 2009

SerpinB2 (aka plasminogen activator type 2) is well described as an extracellular inhibitor of urokinase-type plasminogen activator (uPA). However, the majority of SerpinB2 is retained intracellularly, and many uPA-independent activities have been reported for SerpinB2 suggesting an alternate function. This thesis explores the role of SerpinB2 in epithelial tumour cell lines, highlights the problems associated with various expression systems and argues that SerpinB2 has no role in growth or apoptosis of tumour cells. A potential role for immune modulation and angiogenesis is suggested in in vivo models. Previous research using SerpinB2 transfected, clonally selected tumour cell lines suggested that SerpinB2 regulates the retinoblastoma tumour suppressor protein (Rb) by binding and protecting Rb from degradation. Despite the use of two techniques under numerous conditions and positive controls, no significant interaction between SerpinB2 and Rb was found. SerpinB2 was reported to bind Rb through a PENF homology motif located within the SerpinB2 C-D interhelical loop region. The PENF homology motif was postulated to represent the motif responsible for binding to the C-pocket of Rb. Epstein Barr Virus nuclear antigen 6 (EBNA6) is a known Rb binding protein, which contains two predicted PENF homology motifs. However, mutation of the two PENF homology motifs within EBNA6 did not reduce Rb binding. Furthermore, the SerpinB2 PENF homology motif is actually not well conserved between SerpinB2 proteins from multiple species, whereas other regions of the SerpinB2 C-D loop show a high level of conservation. These data do not support a role for SerpinB2 and the PENF homology motif in Rb binding. SerpinB2 has been proposed to have a role in regulating growth and apoptosis. To further investigate this proposed phenotype of SerpinB2, SerpinB2 was expressed in a range of epithelial tumour lines using transient transfection. No change in growth, apoptosis or Rb levels were found. After ≈2-3 month antibiotic selection for the SerpinB2-expressing plasmid, SerpinB2 protein was lost without the loss of the transgene, indicating selective pressure against long-term SerpinB2 protein expression. To further investigate long-term SerpinB2 expression adenovirus and lentivirus vectors were used. Infection of tumour cell lines with adenovirus vectors expressing SerpinB2 resulted in reduced cell growth, characterised by increased p53 (but not Rb) levels and G2 arrest or apoptosis. When SerpinB2 expressing lentivirus vectors were used to transduce the same tumour cell lines, high levels of long-term expression of functional SerpinB2 was achieved. However, SerpinB2-expressing cell lines showed no differences in growth, proliferation, Rb levels, or apoptosis induced by a range of agents. Growth and apoptosis observed with adenovirus SerpinB2 had all the characteristics of adenovirus-associated toxicity, which has been reported previously for specific proteins. These experiments highlighted the problems associated with SerpinB2 expression systems and suggest that SerpinB2 expression per se is not toxic nor has a role in regulating Rb, growth and apoptosis. Screening of a number of tumour cell lines identified the HPV16 transformed cervical cancer line as expressing high levels of SerpinB2. SerpinB2 was located both extracellularly and intracellularly with a cytoplasmic and nuclear distribution. A high molecular weight SerpinB2 species was identified in CaSki cells and was shown to be the N-linked glycosylated species. Sequencing showed the protein to be Type A SerpinB2 and the protein was shown to form an inhibitory complex with uPA. An abundant low molecular weight SerpinB2 species was also identified in CaSki cell supernatants and appeared to be a proteolytic fragment of SerpinB2. Treatment of CaSki with PMA, TNFα and IFNγ increased SerpinB2 levels. Lentiviral based shRNA failed to significantly down regulate SerpinB2 expression and increasing SerpinB2 levels with lentiviral expression did not change growth, apoptosis, Rb levels or E7 transcription. Lentiviral expression of SerpinB2 in (normally SerpinB2 negative) HPV16 transformed SiHa cells, also failed to show changes in Rb levels or E7 transcription. CaSki thus express wild-type and functional SerpinB2, but no evidence could found that SerpinB2 effects HPV16 E7 transcription or Rb levels. The data presented identifies CaSki as valuable source of biologically functional SerpinB2. SerpinB2 expression in breast cancer cells has been associated with positive prognosis. Tubo, a SerpinB2-negative murine breast carcinoma cell line, was transduced with lentivirus expressing SerpinB2 and grown subcutaneously in BALB/c mice. SerpinB2 expressing tumours appeared red and were larger than control tumours. Furthermore, SerpinB2 expressing tumours had a ≈2 fold higher density of blood vessels when compared to Tubo and Tubo expressing EGFP. Mice carrying tumours expressing SerpinB2 also showed reduced anti-tumour IgG2 responses. These data suggest that a role for SerpinB2 in regulating angiogenesis and antitumour immunity. In conclusion, this thesis challenges the notion that SerpinB2 regulates Rb, cell cycle, and apoptosis and suggests a potential role for SerpinB2 in tumour angiogenesis and immunity.

With global freshwater supplies under pressure, viable water reuse methods are being examined to assist in improving water supplies. Municipal effluent is an ideal source for water reclamation as it is consistent in quality and quantity. The health aspects of water reuse have been identified as an issue of concern, in particular the potential presence of enteric viruses. Managed Aquifer Recharge (MAR) is a method that can aid water reclamation by recharging water such as treated effluent into a suitable aquifer. Research into the removal of pathogenic contaminants by natural processes within aquifers, namely the action of autochthonous bacteria, has led to the consideration that MAR could be used to assist in the removal of microbial pathogens. Pathogens have been demonstrated to be removed during residence in groundwater, but the presence of active autochthonous groundwater bacteria are required for significant removal rates to occur. The aim of this research was to investigate the interaction between autochthonous groundwater bacteria (AGB) and the enteroviruses Poliovirus type 1, Coxsackievirus B3 and Adenovirus B41. It was established that these viruses decrease in number in the presence of AGB but the mechanisms causing this decrease are poorly understood. Experiments were designed to examine how the individual AGB caused decay of the viruses. In this study AGB were isolated and tested for their ability in increase the decay of the viruses. It was determined that 27 % (17/63) of the isolated AGB influenced viral decay. The AGB isolates varied in their influence with only 3 out of 17 isolates being able to cause of the decay of both poliovirus and coxsackievirus. Similar variations in decay were observed for adenovirus. Decay times for all three viruses varied amongst the AGB and between the viruses. Experiments were undertaken to characterise the mechanism causing the antiviral activity of four groundwater isolates (1G, 3A, 4B and 9G) under varying conditions and treatments to give insight into the compounds or mechanisms responsible for viral decay. This would indicate whether compounds produced by the AGB responsible for viral decay were closely associated to bacterial cells (perhaps membrane bound), independent of metabolic activity, heat labile or were enzymatic in nature. The influence of enzyme inhibiters and heat treatment indicated that viral degradation is caused by compounds that are enzymatic in nature. As viral numbers were monitored by nucleic acid copy numbers rather than via infectivity assays, the viral protein coats must be the first step in degradation followed by the removal of the viral nucleic acid. This two step process would require both protease and nuclease enzymes to result in loss of viral numbers as measured by RT-PCR/PCR. Further characterisation and identification of these four bacterial isolates was also carried out. Three out of the four isolates were sequenced and analysed using partial 16S rRNA gene sequences to determine their phylogenetic relationships compared to related organisms. Isolate 3A was placed in the order Burkholderiales. Isolate 4B was placed in the family Xanthomonadaceae. Isolate 9G was placed in the family Rhizobiaceae. Isolate 1G was only partially sequenced and preliminary identification placed it in the phylum Bacteriodetes. Understanding of the processes carried out by AGB within an aquifer during MAR using reclaimed waters will aid in increasing the viability of this water reuse process. If important natural processes could be utilised to remediate any potential pathogens, the health concerns with reclaimed waters could be addressed and solved simply through prescribed retention times within the aquifer. Key species of AGB may even be utilised as markers to assess the suitability of an aquifer for MAR.

SerpinB2 (aka plasminogen activator type 2) is well described as an extracellular inhibitor of urokinase-type plasminogen activator (uPA). However, the majority of SerpinB2 is retained intracellularly, and many uPA-independent activities have been reported for SerpinB2 suggesting an alternate function. This thesis explores the role of SerpinB2 in epithelial tumour cell lines, highlights the problems associated with various expression systems and argues that SerpinB2 has no role in growth or apoptosis of tumour cells. A potential role for immune modulation and angiogenesis is suggested in in vivo models. Previous research using SerpinB2 transfected, clonally selected tumour cell lines suggested that SerpinB2 regulates the retinoblastoma tumour suppressor protein (Rb) by binding and protecting Rb from degradation. Despite the use of two techniques under numerous conditions and positive controls, no significant interaction between SerpinB2 and Rb was found. SerpinB2 was reported to bind Rb through a PENF homology motif located within the SerpinB2 C-D interhelical loop region. The PENF homology motif was postulated to represent the motif responsible for binding to the C-pocket of Rb. Epstein Barr Virus nuclear antigen 6 (EBNA6) is a known Rb binding protein, which contains two predicted PENF homology motifs. However, mutation of the two PENF homology motifs within EBNA6 did not reduce Rb binding. Furthermore, the SerpinB2 PENF homology motif is actually not well conserved between SerpinB2 proteins from multiple species, whereas other regions of the SerpinB2 C-D loop show a high level of conservation. These data do not support a role for SerpinB2 and the PENF homology motif in Rb binding. SerpinB2 has been proposed to have a role in regulating growth and apoptosis. To further investigate this proposed phenotype of SerpinB2, SerpinB2 was expressed in a range of epithelial tumour lines using transient transfection. No change in growth, apoptosis or Rb levels were found. After ≈2-3 month antibiotic selection for the SerpinB2-expressing plasmid, SerpinB2 protein was lost without the loss of the transgene, indicating selective pressure against long-term SerpinB2 protein expression. To further investigate long-term SerpinB2 expression adenovirus and lentivirus vectors were used. Infection of tumour cell lines with adenovirus vectors expressing SerpinB2 resulted in reduced cell growth, characterised by increased p53 (but not Rb) levels and G2 arrest or apoptosis. When SerpinB2 expressing lentivirus vectors were used to transduce the same tumour cell lines, high levels of long-term expression of functional SerpinB2 was achieved. However, SerpinB2-expressing cell lines showed no differences in growth, proliferation, Rb levels, or apoptosis induced by a range of agents. Growth and apoptosis observed with adenovirus SerpinB2 had all the characteristics of adenovirus-associated toxicity, which has been reported previously for specific proteins. These experiments highlighted the problems associated with SerpinB2 expression systems and suggest that SerpinB2 expression per se is not toxic nor has a role in regulating Rb, growth and apoptosis. Screening of a number of tumour cell lines identified the HPV16 transformed cervical cancer line as expressing high levels of SerpinB2. SerpinB2 was located both extracellularly and intracellularly with a cytoplasmic and nuclear distribution. A high molecular weight SerpinB2 species was identified in CaSki cells and was shown to be the N-linked glycosylated species. Sequencing showed the protein to be Type A SerpinB2 and the protein was shown to form an inhibitory complex with uPA. An abundant low molecular weight SerpinB2 species was also identified in CaSki cell supernatants and appeared to be a proteolytic fragment of SerpinB2. Treatment of CaSki with PMA, TNFα and IFNγ increased SerpinB2 levels. Lentiviral based shRNA failed to significantly down regulate SerpinB2 expression and increasing SerpinB2 levels with lentiviral expression did not change growth, apoptosis, Rb levels or E7 transcription. Lentiviral expression of SerpinB2 in (normally SerpinB2 negative) HPV16 transformed SiHa cells, also failed to show changes in Rb levels or E7 transcription. CaSki thus express wild-type and functional SerpinB2, but no evidence could found that SerpinB2 effects HPV16 E7 transcription or Rb levels. The data presented identifies CaSki as valuable source of biologically functional SerpinB2. SerpinB2 expression in breast cancer cells has been associated with positive prognosis. Tubo, a SerpinB2-negative murine breast carcinoma cell line, was transduced with lentivirus expressing SerpinB2 and grown subcutaneously in BALB/c mice. SerpinB2 expressing tumours appeared red and were larger than control tumours. Furthermore, SerpinB2 expressing tumours had a ≈2 fold higher density of blood vessels when compared to Tubo and Tubo expressing EGFP. Mice carrying tumours expressing SerpinB2 also showed reduced anti-tumour IgG2 responses. These data suggest that a role for SerpinB2 in regulating angiogenesis and antitumour immunity. In conclusion, this thesis challenges the notion that SerpinB2 regulates Rb, cell cycle, and apoptosis and suggests a potential role for SerpinB2 in tumour angiogenesis and immunity.

SerpinB2 (aka plasminogen activator type 2) is well described as an extracellular inhibitor of urokinase-type plasminogen activator (uPA). However, the majority of SerpinB2 is retained intracellularly, and many uPA-independent activities have been reported for SerpinB2 suggesting an alternate function. This thesis explores the role of SerpinB2 in epithelial tumour cell lines, highlights the problems associated with various expression systems and argues that SerpinB2 has no role in growth or apoptosis of tumour cells. A potential role for immune modulation and angiogenesis is suggested in in vivo models. Previous research using SerpinB2 transfected, clonally selected tumour cell lines suggested that SerpinB2 regulates the retinoblastoma tumour suppressor protein (Rb) by binding and protecting Rb from degradation. Despite the use of two techniques under numerous conditions and positive controls, no significant interaction between SerpinB2 and Rb was found. SerpinB2 was reported to bind Rb through a PENF homology motif located within the SerpinB2 C-D interhelical loop region. The PENF homology motif was postulated to represent the motif responsible for binding to the C-pocket of Rb. Epstein Barr Virus nuclear antigen 6 (EBNA6) is a known Rb binding protein, which contains two predicted PENF homology motifs. However, mutation of the two PENF homology motifs within EBNA6 did not reduce Rb binding. Furthermore, the SerpinB2 PENF homology motif is actually not well conserved between SerpinB2 proteins from multiple species, whereas other regions of the SerpinB2 C-D loop show a high level of conservation. These data do not support a role for SerpinB2 and the PENF homology motif in Rb binding. SerpinB2 has been proposed to have a role in regulating growth and apoptosis. To further investigate this proposed phenotype of SerpinB2, SerpinB2 was expressed in a range of epithelial tumour lines using transient transfection. No change in growth, apoptosis or Rb levels were found. After ≈2-3 month antibiotic selection for the SerpinB2-expressing plasmid, SerpinB2 protein was lost without the loss of the transgene, indicating selective pressure against long-term SerpinB2 protein expression. To further investigate long-term SerpinB2 expression adenovirus and lentivirus vectors were used. Infection of tumour cell lines with adenovirus vectors expressing SerpinB2 resulted in reduced cell growth, characterised by increased p53 (but not Rb) levels and G2 arrest or apoptosis. When SerpinB2 expressing lentivirus vectors were used to transduce the same tumour cell lines, high levels of long-term expression of functional SerpinB2 was achieved. However, SerpinB2-expressing cell lines showed no differences in growth, proliferation, Rb levels, or apoptosis induced by a range of agents. Growth and apoptosis observed with adenovirus SerpinB2 had all the characteristics of adenovirus-associated toxicity, which has been reported previously for specific proteins. These experiments highlighted the problems associated with SerpinB2 expression systems and suggest that SerpinB2 expression per se is not toxic nor has a role in regulating Rb, growth and apoptosis. Screening of a number of tumour cell lines identified the HPV16 transformed cervical cancer line as expressing high levels of SerpinB2. SerpinB2 was located both extracellularly and intracellularly with a cytoplasmic and nuclear distribution. A high molecular weight SerpinB2 species was identified in CaSki cells and was shown to be the N-linked glycosylated species. Sequencing showed the protein to be Type A SerpinB2 and the protein was shown to form an inhibitory complex with uPA. An abundant low molecular weight SerpinB2 species was also identified in CaSki cell supernatants and appeared to be a proteolytic fragment of SerpinB2. Treatment of CaSki with PMA, TNFα and IFNγ increased SerpinB2 levels. Lentiviral based shRNA failed to significantly down regulate SerpinB2 expression and increasing SerpinB2 levels with lentiviral expression did not change growth, apoptosis, Rb levels or E7 transcription. Lentiviral expression of SerpinB2 in (normally SerpinB2 negative) HPV16 transformed SiHa cells, also failed to show changes in Rb levels or E7 transcription. CaSki thus express wild-type and functional SerpinB2, but no evidence could found that SerpinB2 effects HPV16 E7 transcription or Rb levels. The data presented identifies CaSki as valuable source of biologically functional SerpinB2. SerpinB2 expression in breast cancer cells has been associated with positive prognosis. Tubo, a SerpinB2-negative murine breast carcinoma cell line, was transduced with lentivirus expressing SerpinB2 and grown subcutaneously in BALB/c mice. SerpinB2 expressing tumours appeared red and were larger than control tumours. Furthermore, SerpinB2 expressing tumours had a ≈2 fold higher density of blood vessels when compared to Tubo and Tubo expressing EGFP. Mice carrying tumours expressing SerpinB2 also showed reduced anti-tumour IgG2 responses. These data suggest that a role for SerpinB2 in regulating angiogenesis and antitumour immunity. In conclusion, this thesis challenges the notion that SerpinB2 regulates Rb, cell cycle, and apoptosis and suggests a potential role for SerpinB2 in tumour angiogenesis and immunity.

Thesis (Ph.D. in Immunology) -- University of Colorado at Denver and Health Sciences Center, 2006. / Typescript. Includes bibliographical references (leaves 129-141). Free to UCDHSC affiliates. Online version available via ProQuest Digital Dissertations;

Diss. (sammanfattning) Uppsala : Univ., 2001. / Härtill 3 uppsatser.