Academic literature on the topic 'Antigen variation study'
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Journal articles on the topic "Antigen variation study"
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Röske, Kerstin, Alain Blanchard, Isabelle Chambaud, Christine Citti, Jürgen H. Helbig, Marie-Christine Prevost, Renate Rosengarten, and Enno Jacobs. "Phase Variation among Major Surface Antigens ofMycoplasma penetrans." Infection and Immunity 69, no. 12 (December 1, 2001): 7642–51. http://dx.doi.org/10.1128/iai.69.12.7642-7651.2001.
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ABSTRACT The pathogenicity and prevalence of Mycoplasma penetrans, a Mycoplasma species recently isolated from humans, are still debated. A major P35 antigen, which is used as target epitope in serological assays, was shown to be a phase-variable lipid-associated membrane protein (LAMP). In this study, we performed a comparative analysis of the LAMP patterns from fiveM. penetrans clinical isolates and from the type strain. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis profiles and immunoblots with sera serially collected from an M. penetrans-infected patient indicated that these strains expressed different LAMP repertoires. Furthermore, the intraclonal variation in the expression of LAMPs (P34A, P34B, P35, and P38) was monitored by immunoblot analysis with three specific monoclonal antibodies (MAbs) developed in this study and MAb 7 to P35. The phase variation of these LAMPs occurs in an independent manner, with frequencies of variation ranging from 10−2 to 10−4 per cell per generation. Consistent with their amphipathic nature, the P34B and P38 antigens were found exposed at the cell surface. The DNA sequence encoding the P38 antigen was defined and found to be related to those of the P35 gene and other putative LAMP-encoding genes, suggesting that these variable antigens are encoded by a family of related genes. Finally, the serum samples from an M. penetrans-infected patient contained antibodies that reacted with a P36 antigen expressed in different M. penetrans strains but not in the isolate recovered from this patient. This result suggested that in vivo phase variation of P36 occurred, which would support a role for these LAMP variations in avoiding the host's immune vigilance.
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Beem, J. E., W. B. Clark, and A. S. Bleiweis. "Antigenic Variation of Indigenous Streptococci." Journal of Dental Research 64, no. 8 (August 1985): 1039–45. http://dx.doi.org/10.1177/00220345850640080301.
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Isolates of Group D streptococci indigenous to the murine oral cavity were studied to detect the occurrence of antigenic variation. Group D streptococci cultured from molar homogenates of Balb/c mice were randomly selected for study on the basis of distinctive colony morphology. Isolates obtained over a 12-week period were biotyped using the API 20S system, and subjected to Lancefield extraction and rocket immunoelectrophoresis for serotyping. All isolates were compared with an arbitrarily selected standard test strain (W1S-1) isolated the first week of the first experimental series. Four biotypes were encountered during the first week of two experimental series. Two very unusual biotypes detected during the first experimental series persisted throughout that series, as did two more common biotypes throughout the second experimental series. Anti-W1S-1 serum produced three precipitin bands (antigens O, D, and K) against WIS-1 Lancefield extract and against the respective biotypes detected during the first week of the two series. Of the three antigens detected, only the group antigen (D) did not vary during either experimental series. Antigenic variants lacking the O or K antigen and bearing these distinctive phenotypes were repeatedly isolated in subsequent weeks. Ultimately, 16% of 190 strains isolated during the first series and 26% of 167 strains isolated during the second series proved to be antigenic variants of the predominant biotypes detected in both series.
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Li, Qun, Matthew Hobbs, and Peter R. Reeves. "The variation of dTDP-l-rhamnose pathway genes in Vibrio cholerae." Microbiology 149, no. 9 (September 1, 2003): 2463–74. http://dx.doi.org/10.1099/mic.0.26382-0.
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The genetic variation in the dTDP-l-rhamnose pathway genes (rmlA, rmlB, rmlC and rmlD) in Vibrio cholerae was investigated. The genes are part of the O antigen gene cluster and the aim was to study lateral gene transfer of O antigen gene clusters. The rml genes of an O6 strain were cloned using an Escherichia coli K-12 strain designed for selecting cloned rml genes. Thirty-three strains carrying the known rhamnose-containing O antigens were probed with O6-based rml gene probes, and 19 were positive with from one to all four of the gene probes. Nine rml gene sets from this group were sequenced and found to be in the order rmlBADC, at the 5′ end of the gene clusters. A gradient in the level of variation was observed, with highly similar sequences at the 5′ end rmlB gene, but very divergent and strain-specific sequences at the 3′ end of the rml gene set. The change in level of similarity varied in position, but was always abrupt and coincided with a change in GC content, indicating that the 5′ and 3′ parts are of different origin, and that recombination within rml genes has occurred. The rml gene sets of two of the strains that did not hybridize with any O6 rml gene probes were also cloned and sequenced. Both gene sets were in the middle of the O antigen gene cluster and were very divergent from each other and all other rml gene sets. This supports the hypothesis that presence of rml genes at the end of the O antigen gene cluster facilitates lateral gene transfer of rml-containing O antigen gene clusters in V. cholerae. The sequence relationships make it possible to identify sites of recombination and to distinguish DNA that has long been in V. cholerae and DNA that probably came into the species with the O antigen gene cluster.
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IGLESIAS, R., A. PARAMÁ, M. F. ÁLVAREZ, J. LEIRO, F. M. UBEIRA, and M. L. SANMARTÍN. "Philasterides dicentrarchi (Ciliophora: Scuticociliatida) expresses surface immobilization antigens that probably induce protective immune responses in turbot." Parasitology 126, no. 2 (February 2003): 125–34. http://dx.doi.org/10.1017/s0031182002002688.
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Philasterides dicentrarchi is a histophagous ciliate causing systemic scuticociliatosis in cultured turbot. This study demonstrates that turbot which survive this disease have serum antibodies that recognize ciliary antigens of this ciliate in ELISA and immobilize/agglutinate the ciliate in vitro. Mouse sera raised against ciliary antigens and integral membrane proteins are likewise capable of immobilizing/agglutinating the ciliates, indicating that P. dicentrarchi, like other ciliates, expresses surface immobilization antigens. Furthermore, the antigen agglutinating reaction induces the parasite to shed its surface antigens rapidly, replacing them with others with different specific serology. This antigen shedding and variation response is similar to that detected in other protozoan parasites. Immunization of turbot with ciliate lysate plus adjuvant or with formalin-fixed ciliates induced synthesis of agglutinating antibodies and conferred a degree of protection against challenge infection, suggesting that the response to surface antigens may play an important role in defence against this pathogen, SDS–PAGE and immunoblotting studies indicated the existence of a predominant polypeptide of about 38 kDa in the ciliary antigen and membrane protein fractions, and this may be the principal surface antigen of P. dicentrarchi.
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Bertina, R. M. "An International Collaborative Study on the Performance of Protein C Antigen Assays." Thrombosis and Haemostasis 57, no. 01 (1987): 112–17. http://dx.doi.org/10.1055/s-0038-1651074.
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SummaryAn international collaborative study was undertaken to evaluate the performance and specificity of protein C antigen (PC) assays. Thirteen lyophilized plasma samples were distributed among 17 laboratories and analysed with 24 methods. No statistically significant results were obtained with the different methods in plasmas containing only the protein C zymogen. ELISA’s, RIA’s and IRMA’s were found to be more sensitive than the electro-immunoassay. In plasmas of patients on oral anticoagulant treatment ELISA methods tend to give lower PC antigen levels than the electro-immunoassay. Complexes between activated protein C (APC) and the protein C inhibitor (PCI), when present in plasma together with PC zymogen, are detected with 100% efficiency in the electro-immunoassay, with 50% efficiency in the ELISA, and with <10% efficiency in the RIA or in assays using monoclonal antibodies against PC.Mean coefficient of variation was calculated to be 22%, and could be reduced - especially in case of the ELISA by normalisation. Within laboratory variation was calculated to be 11.7% and between laboratory variation 17.8%.
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Marcotuillo, Michelle, Vicki Johnson, Richard J. Jenny, and Ryan H. Dorfman. "Murine Reagents and Analytical Tools to Enhance the Utility of Mouse Models for the Study of Human Thrombotic Disorders and Disease States." Blood 104, no. 11 (November 16, 2004): 3989. http://dx.doi.org/10.1182/blood.v104.11.3989.3989.
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Abstract The mechanisms that govern blood coagulation and fibrinolysis have been studied extensively by employing a variety of experimental techniques and in-vivo model systems. Murine models are a valuable resource to test new strategies in the areas of thrombosis and haemostasis. Because both normal and genetically altered mice are available, the murine models are ideal for this application. Murine models are in prevalent use and there exists a limited number of reagents available to assist the researcher. Development of murine reagents and analytical tools will enhance the utility of murine coagulation models by allowing researchers to acquire appropriate standards for characterization, validation, and screening of murine model systems. Recently, we have purified and characterized a group of nine mouse plasma proteins including the enzymatic forms of many of these proteins. A total of eight new polyclonal antibodies to murine antigens were generated including sheep a-mouse plasminogen and sheep anti-mouse factor X. Here we report on the development of two competitive based ELISA’s for the quantitation of mouse plasminogen and mouse factor X. The assay format utilizes purified antigen (plasminogen and factor X) standard. A standard curve is generated based upon the competition between fluid-phase antigen and an antigen-biotin conjugate for an immobilized affinity purified polyclonal antibody (sheep a-mouse plasminogen and sheep anti-mouse factor X). Solid-phase antibody/biotinyl-antigen complexes are detected using avidin-peroxidase. A colormetric signal, which is inversely proportional to the amount of antigen in the fluid-phase, is obtained following the addition of chromogenic substrate. Calibration curves are generated for an immobilized antibody ELISA by plotting the absorbance at 490 nm versus the concentration of non-biotin labeled antigen standard. Biotinylated-antigens were used at single concentrations of 50 ng/ml and 10 ng/ml for factor X and plasminogen respectively. In both cases, immobilized antibodies were adsorbed to microtiter plates at 5ug/ ml. Linearity of the factor X assay extends from approximately 10 ng/ml to 1000 ng/ml. The intra-assay coefficient of variation (CV) is ≤1.1 %, while the inter-assay CV is ≤5 %. Linearity of the plasminogen assay extends from approximately 0.1 μg/ml to 10 μg/ml. The intra-assay coefficient of variation (CV) is ≤2.0 %, while the inter-assay CV is ≤6.3 %. Preliminary studies have demonstrated the feasibility of these assays. With further testing and analysis these assays may prove useful to the researcher attempting to quantitate mouse plasma concentrations of these analytes.
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Thornton, Emily E., Mark R. Looney, Oishee Bose, Debasish Sen, Dean Sheppard, Richard Locksley, Xiaozhu Huang, and Matthew F. Krummel. "Spatiotemporally separated antigen uptake by alveolar dendritic cells and airway presentation to T cells in the lung." Journal of Experimental Medicine 209, no. 6 (May 14, 2012): 1183–99. http://dx.doi.org/10.1084/jem.20112667.
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Asthma pathogenesis is focused around conducting airways. The reasons for this focus have been unclear because it has not been possible to track the sites and timing of antigen uptake or subsequent antigen presentation to effector T cells. In this study, we use two-photon microscopy of the lung parenchyma and note accumulation of CD11b+ dendritic cells (DCs) around the airway after allergen challenge but very limited access of these airway-adjacent DCs to the contents of the airspace. In contrast, we observed prevalent transepithelial uptake of particulate antigens by alveolar DCs. These distinct sites are temporally linked, as early antigen uptake in alveoli gives rise to DC and antigen retention in the airway-adjacent region. Antigen-specific T cells also accumulate in the airway-adjacent region after allergen challenge and are activated by the accumulated DCs. Thus, we propose that later airway hyperreactivity results from selective retention of allergen-presenting DCs and antigen-specific T cells in airway-adjacent interaction zones, not from variation in the abilities of individual DCs to survey the lung.
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Saeed, Mohd, Vikas Kushwaha, Syed Mohd Faisal, Richa Verma, Irfan Ahmad, Huma Mustafa, Magdah Ganash, Mohammad Amjad Kamal, and Ghulam Md Ashraf. "A Study on Serological Reactivity Profile of Different Antigen Preparations with Bancroftian filariasis Human Infection Sera." Protein & Peptide Letters 27, no. 9 (October 15, 2020): 841–50. http://dx.doi.org/10.2174/0929866527666200225123534.
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Background: Lymphatic Filariasis (LF) is one of the incapacitating and mosquito-borne sicknesses that on progression may prompt a few recognizable types of clutters like extreme lymphedema, hydrocele, and elephantiasis. Methods: Antigenic preparations of B. malayi adult (BmA), S. cervi adult parasites and microfilariae (mf) total parasite extract were used to analyze the serological reactivity profile with human infectious sera collected from endemic areas of Bancroftian filariasis by performing Western blot and ELISA analysis. Sera from healthy human subjects were also included in the study to determine the variation incurred in the reactivity due to the filariasis infection. Gelelectrophoresis analysis of the crude-extract of BmA revealed seven protein bands while more than ten bands were recognized in S. cervi. Results: our results represent a clear variation in protein patterns among the crude-antigens. ELISA results showed highest prevalence of IgG, IgM and IgG4 antibodies against all antigen preparations when recorded among microfilaraemic chronic infected patients. In both the antigenic preparations, the positive reactions were in the order of microfilaraemic>endemic normal>chronic>acute>nonendemic normal subjects. All sera of Mf+ patients were uniformly positive, while sera of both chronic and endemic normal subjects showed less reactivity. Conclusion: In the present study, we endeavoured to establish the extent of cross-reactivity of antigens derived from animal filarial parasites such as B. malayi and S. cervi with W. bancrofti filariasis sera of human patients. Besides, we further analyzed antibody-isotype profile of IgG, IgG4 and IgM in various human infection sera of bancroftian filarial subjects reactive to heterologous parasite antigens derived from adult worms of S. cervi from bovine and B. malayi from bovine and jirds.
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Horino, Atsuko, Yuko Sasaki, Tsuguo Sasaki, and Tsuyoshi Kenri. "Multiple Promoter Inversions Generate Surface Antigenic Variation in Mycoplasma penetrans." Journal of Bacteriology 185, no. 1 (January 1, 2003): 231–42. http://dx.doi.org/10.1128/jb.185.1.231-242.2003.
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ABSTRACT Mycoplasma penetrans is a newly identified species of the genus Mycoplasma. It was first isolated from a urine sample from a human immunodeficiency virus (HIV)-infected patient. M. penetrans changes its surface antigen profile with high frequency. The changes originate from ON↔OFF phase variations of the P35 family of surface membrane lipoproteins. The P35 family lipoproteins are major antigens recognized by the human immune system during M. penetrans infection and are encoded by the mpl genes. Phase variations of P35 family lipoproteins occur at the transcriptional level of mpl genes; however, the precise genetic mechanisms are unknown. In this study, the molecular mechanisms of surface antigen profile change in M. penetrans were investigated. The focus was on the 46-kDa protein that is present in M. penetrans strain HF-2 but not in the type strain, GTU. The 46-kDa protein was the product of a previously reported mpl gene, pepIMP13, with an amino-terminal sequence identical to that of the P35 family lipoproteins. Nucleotide sequencing analysis of the pepIMP13 gene region revealed that the promoter-containing 135-bp DNA of this gene had the structure of an invertible element that functioned as a switch for gene expression. In addition, all of the mpl genes of M. penetrans HF-2 were identified using the whole-genome sequence data that has recently become available for this bacterium. There are at least 38 mpl genes in the M. penetrans HF-2 genome. Interestingly, most of these mpl genes possess invertible promoter-like sequences, similar to those of the pepIMP13 gene promoter. A model for the generation of surface antigenic variation by multiple promoter inversions is proposed.
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Umezawa, Eufrosina S., Sueli F. Bastos, Mario E. Camargo, Luci M. Yamauchi, Márcia R. Santos, Antonio Gonzalez, Bianca Zingales, et al. "Evaluation of Recombinant Antigens for Serodiagnosis of Chagas’ Disease in South and Central America." Journal of Clinical Microbiology 37, no. 5 (1999): 1554–60. http://dx.doi.org/10.1128/jcm.37.5.1554-1560.1999.
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The commercially available diagnostic tests for Chagas’ disease employ whole extracts or semipurified fractions ofTrypanosoma cruzi epimastigotes. Considerable variation in the reproducibility and reliability of these tests has been reported by different research laboratories, mainly due to cross-reactivity with other pathogens and standardization of the reagents. The use of recombinant antigens for the serodiagnosis of Chagas’ disease is recommended to increase the sensitivity and specificity of serological tests. Expressed in Escherichia coli, as fusion products with glutathione S-transferase, six T. cruzirecombinant antigens (H49, JL7, A13, B13, JL8, and 1F8) were evaluated in an enzyme-linked immunosorbent assay for Chagas’ disease. The study was carried out with a panel of 541 serum samples of chagasic and nonchagasic patients from nine countries of Latin America (Argentina, Bolivia, Brazil, Chile, Colombia, El Salvador, Guatemala, Honduras, and Venezuela). The optimal concentration of each recombinant antigen for coating of plates was determined with the help of125I-labelled recombinant proteins. While the specificity of the epimastigote antigen was 84% because of false positives from leishmaniasis cases, for the recombinant antigens it varied from 96.2 to 99.6%. Recombinant antigens reacted with 79 to 100% of serum samples from chronic chagasic patients. In this way, it is proposed that a mixture of a few T. cruzi recombinant antigens should be employed in a diagnostic kit to minimize individual variation and promote high sensitivity in the diagnosis of Chagas’ disease.
Dissertations / Theses on the topic "Antigen variation study"
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Srivastava, Sanjeev Kumar. "MHC class II antigen variation study in selected populations of Northern parts of India." Thesis, University of North Bengal, 2007. http://hdl.handle.net/123456789/1011.
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Yong, Patrick. "A study of the variation of leukocyte immunoglobulin-like receptors on antigen-presenting cells." Thesis, King's College London (University of London), 2013. https://kclpure.kcl.ac.uk/portal/en/theses/a-study-of-the-variation-of-leukocyte-immunoglobulin-like-receptors-on-antigen-presenting-cells(a79cecd9-abf2-4828-a70d-29c3dcc797e4).html.
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Leukocyte immunoglobulin-like receptors are a family of inhibitory and activating receptors found on a wide variety of immune cells, and are thought to play a significant role in determining immune responses. Their known ligands are HLA Class I molecules; HLA-G is the prototypic ligand which has high affinity for two inhibitory members of the family and mediates its immunosuppressive functions through them. The hypothesis of this study is that polymorphisms in the non-coding regions are likely to influence both the expression and response of the LILRs to various stimuli. Published data supports this view in that polymorphisms in the LILRs are associated with various immunologically mediated diseases. In addition, these molecules represent a potential target for therapeutic manipulation and attempts to develop a therapy using this route were undertaken. Expression levels of various LILR molecules on APCs at baseline and after stimulation have shown significant variation between subjects. Genomic variation in the promoter regions of LILRB2 was established by sequencing. The relationship between LILRB2 SNPs and cell expression levels was tested both directly and in a luciferase assay, but no significant associations were found. Engagement of LILRB2 by monoclonal antibodies was shown to affect TLR-induced cytokine secretion, but no relationship was found between LILRB2 expression levels and the effect on cytokine secretion. A synthetic construct (the “G-body”) utilising the LILR-HLA-G interaction has been developed for testing as a potential therapeutic molecule. This construct comprises two functional domains – an anti-HLA class I domain and an HLA-G domain. This construct would be expected to modulate allogeneic responses by localisation of an immunosuppressive signal (HLA-G) to the allogeneic HLA molecule; and (ii) potentially masking the allogeneic HLA class I molecule. Testing has shown that the construct suppresses lymphocyte proliferation with enhancement of this effect dependent on the presence of allogeneic HLA class I.
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Graf, Justin T. "Membrane associated transporter protein gene (SLC45A2) and the genetic basis of normal human pigmentation variation." Thesis, Queensland University of Technology, 2008. https://eprints.qut.edu.au/25913/1/Justin_Graf_Thesis.pdf.
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This work is concerned with the genetic basis of normal human pigmentation variation. Specifically, the role of polymorphisms within the solute carrier family 45 member 2 (SLC45A2 or membrane associated transporter protein; MATP) gene were investigated with respect to variation in hair, skin and eye colour ― both between and within populations. SLC45A2 is an important regulator of melanin production and mutations in the gene underly the most recently identified form of oculocutaneous albinism. There is evidence to suggest that non-synonymous polymorphisms in SLC45A2 are associated with normal pigmentation variation between populations. Therefore, the underlying hypothesis of this thesis is that polymorphisms in SLC45A2 will alter the function or regulation of the protein, thereby altering the important role it plays in melanogenesis and providing a mechanism for normal pigmentation variation.
In order to investigate the role that SLC45A2 polymorphisms play in human pigmentation variation, a DNA database was established which collected pigmentation phenotypic information and blood samples of more than 700 individuals. This database was used as the foundation for two association studies outlined in this thesis, the first of which involved genotyping two previously-described non-synonymous polymorphisms, p.Glu272Lys and p.Phe374Leu, in four different population groups. For both polymorphisms, allele frequencies were significantly different between population groups and the 272Lys and 374Leu alleles were strongly associated with black hair, brown eyes and olive skin colour in Caucasians. This was the first report to show that SLC45A2 polymorphisms were associated with normal human intra-population pigmentation variation.
The second association study involved genotyping several SLC45A2 promoter polymorphisms to determine if they also played a role in pigmentation variation. Firstly, the transcription start site (TSS), and hence putative proximal promoter region, was identified using 5' RNA ligase mediated rapid amplification of cDNA ends (RLM-RACE). Two alternate TSSs were identified and the putative promoter region was screened for novel polymorphisms using denaturing high performance liquid chromatography (dHPLC). A novel duplication (c.–1176_–1174dupAAT) was identified along with other previously described single nucleotide polymorphisms (c.–1721C>G and c.–1169G>A). Strong linkage disequilibrium ensured that all three polymorphisms were associated with skin colour such that the –1721G, +dup and –1169A alleles were associated with olive skin in Caucasians. No linkage disequilibrium was observed between the promoter and coding region polymorphisms, suggesting independent effects. The association analyses were complemented with functional data, showing that the –1721G, +dup and –1169A alleles significantly decreased SLC45A2 transcriptional activity. Based on in silico bioinformatic analysis that showed these alleles remove a microphthalmia-associated transcription factor (MITF) binding site, and that MITF is a known regulator of SLC45A2 (Baxter and Pavan, 2002; Du and Fisher, 2002), it was postulated that SLC45A2 promoter polymorphisms could contribute to the regulation of pigmentation by altering MITF binding affinity.
Further characterisation of the SLC45A2 promoter was carried out using luciferase reporter assays to determine the transcriptional activity of different regions of the promoter. Five constructs were designed of increasing length and their promoter activity evaluated. Constitutive promoter activity was observed within the first ~200 bp and promoter activity increased as the construct size increased. The functional impact of the –1721G, +dup and –1169A alleles, which removed a MITF consensus binding site, were assessed using electrophoretic mobility shift assays (EMSA) and expression analysis of genotyped melanoblast and melanocyte cell lines. EMSA results confirmed that the promoter polymorphisms affected DNA-protein binding. Interestingly, however, the protein/s involved were not MITF, or at least MITF was not the protein directly binding to the DNA. In an effort to more thoroughly characterise the functional consequences of SLC45A2 promoter polymorphisms, the mRNA expression levels of SLC45A2 and MITF were determined in melanocyte/melanoblast cell lines. Based on SLC45A2’s role in processing and trafficking TYRP1 from the trans-Golgi network to stage 2 melanosmes, the mRNA expression of TYRP1 was also investigated. Expression results suggested a coordinated expression of pigmentation genes.
This thesis has substantially contributed to the field of pigmentation by showing that SLC45A2 polymorphisms not only show allele frequency differences between population groups, but also contribute to normal pigmentation variation within a Caucasian population. In addition, promoter polymorphisms have been shown to have functional consequences for SLC45A2 transcription and the expression of other pigmentation genes. Combined, the data presented in this work supports the notion that SLC45A2 is an important contributor to normal pigmentation variation and should be the target of further research to elucidate its role in determining pigmentation phenotypes. Understanding SLC45A2’s function may lead to the development of therapeutic interventions for oculocutaneous albinism and other disorders of pigmentation. It may also help in our understanding of skin cancer susceptibility and evolutionary adaptation to different UV environments, and contribute to the forensic application of pigmentation phenotype prediction.
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Graf, Justin T. "Membrane associated transporter protein gene (SLC45A2) and the genetic basis of normal human pigmentation variation." Queensland University of Technology, 2008. http://eprints.qut.edu.au/25913/.
Full textAbstract:
This work is concerned with the genetic basis of normal human pigmentation variation. Specifically, the role of polymorphisms within the solute carrier family 45 member 2 (SLC45A2 or membrane associated transporter protein; MATP) gene were investigated with respect to variation in hair, skin and eye colour ― both between and within populations. SLC45A2 is an important regulator of melanin production and mutations in the gene underly the most recently identified form of oculocutaneous albinism. There is evidence to suggest that non-synonymous polymorphisms in SLC45A2 are associated with normal pigmentation variation between populations. Therefore, the underlying hypothesis of this thesis is that polymorphisms in SLC45A2 will alter the function or regulation of the protein, thereby altering the important role it plays in melanogenesis and providing a mechanism for normal pigmentation variation.
In order to investigate the role that SLC45A2 polymorphisms play in human pigmentation variation, a DNA database was established which collected pigmentation phenotypic information and blood samples of more than 700 individuals. This database was used as the foundation for two association studies outlined in this thesis, the first of which involved genotyping two previously-described non-synonymous polymorphisms, p.Glu272Lys and p.Phe374Leu, in four different population groups. For both polymorphisms, allele frequencies were significantly different between population groups and the 272Lys and 374Leu alleles were strongly associated with black hair, brown eyes and olive skin colour in Caucasians. This was the first report to show that SLC45A2 polymorphisms were associated with normal human intra-population pigmentation variation.
The second association study involved genotyping several SLC45A2 promoter polymorphisms to determine if they also played a role in pigmentation variation. Firstly, the transcription start site (TSS), and hence putative proximal promoter region, was identified using 5' RNA ligase mediated rapid amplification of cDNA ends (RLM-RACE). Two alternate TSSs were identified and the putative promoter region was screened for novel polymorphisms using denaturing high performance liquid chromatography (dHPLC). A novel duplication (c.–1176_–1174dupAAT) was identified along with other previously described single nucleotide polymorphisms (c.–1721C>G and c.–1169G>A). Strong linkage disequilibrium ensured that all three polymorphisms were associated with skin colour such that the –1721G, +dup and –1169A alleles were associated with olive skin in Caucasians. No linkage disequilibrium was observed between the promoter and coding region polymorphisms, suggesting independent effects. The association analyses were complemented with functional data, showing that the –1721G, +dup and –1169A alleles significantly decreased SLC45A2 transcriptional activity. Based on in silico bioinformatic analysis that showed these alleles remove a microphthalmia-associated transcription factor (MITF) binding site, and that MITF is a known regulator of SLC45A2 (Baxter and Pavan, 2002; Du and Fisher, 2002), it was postulated that SLC45A2 promoter polymorphisms could contribute to the regulation of pigmentation by altering MITF binding affinity.
Further characterisation of the SLC45A2 promoter was carried out using luciferase reporter assays to determine the transcriptional activity of different regions of the promoter. Five constructs were designed of increasing length and their promoter activity evaluated. Constitutive promoter activity was observed within the first ~200 bp and promoter activity increased as the construct size increased. The functional impact of the –1721G, +dup and –1169A alleles, which removed a MITF consensus binding site, were assessed using electrophoretic mobility shift assays (EMSA) and expression analysis of genotyped melanoblast and melanocyte cell lines. EMSA results confirmed that the promoter polymorphisms affected DNA-protein binding. Interestingly, however, the protein/s involved were not MITF, or at least MITF was not the protein directly binding to the DNA. In an effort to more thoroughly characterise the functional consequences of SLC45A2 promoter polymorphisms, the mRNA expression levels of SLC45A2 and MITF were determined in melanocyte/melanoblast cell lines. Based on SLC45A2’s role in processing and trafficking TYRP1 from the trans-Golgi network to stage 2 melanosmes, the mRNA expression of TYRP1 was also investigated. Expression results suggested a coordinated expression of pigmentation genes.
This thesis has substantially contributed to the field of pigmentation by showing that SLC45A2 polymorphisms not only show allele frequency differences between population groups, but also contribute to normal pigmentation variation within a Caucasian population. In addition, promoter polymorphisms have been shown to have functional consequences for SLC45A2 transcription and the expression of other pigmentation genes. Combined, the data presented in this work supports the notion that SLC45A2 is an important contributor to normal pigmentation variation and should be the target of further research to elucidate its role in determining pigmentation phenotypes. Understanding SLC45A2’s function may lead to the development of therapeutic interventions for oculocutaneous albinism and other disorders of pigmentation. It may also help in our understanding of skin cancer susceptibility and evolutionary adaptation to different UV environments, and contribute to the forensic application of pigmentation phenotype prediction.
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Karthigesu, Vassandra Devi. "A study of hepatitis B virus variation and antigenic variants." Thesis, Royal Veterinary College (University of London), 1996. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.309416.
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Torres, Puig Sergi. "Study of a master regulator of recombination in Mycoplasma genitalium." Doctoral thesis, Universitat Autònoma de Barcelona, 2017. http://hdl.handle.net/10803/456208.
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Mycoplasma genitalium és un petit patogen humà causant d’uretritis no gonocòccica, cervicitis i algunes malalties d’inflamació pèlvica. Té un dels genomes més petits trobats en un organisme viu autònom i, per aquest motiu, ha esdevingut un model bacterià d’estudi d’una cèl·lula mínima. A més a més, aquest petit patogen presenta un mecanisme únic de variació antigènica que depèn de la recombinació homòloga entre les adhesines principals i elements repetits del genoma que es troben dispersos al llarg del cromosoma. Tot i que M. genitalium va ser un dels primers microorganismes que fou seqüenciat, es tenen molt poques dades del seu reduït circuit genètic i de la seva regulació gènica. Per altra banda, els mecanismes que regulen el procés de variació antigènica són desconeguts tot i ser essencial per a l’evasió immune i la persistència de la infecció. En aquesta tesi doctoral, hem estudiat el rol de la proteïna MG428 (20), la qual es tracta d’un factor sigma alternatiu d’aquest petit bacteri. La proteïna MG428 reconeix una zona promotora nova i activa la transcripció d’un reguló petit format per gens que codifiquen per enzims de recombinació clau, gens que codifiquen per proteïnes hipotètiques i també regions no codificants de funció desconeguda. A més a més, anàlisis de cèl·lules individuals han confirmat que l’activació via 20 només té lloc en un percentatge petit de la població al mateix temps i també mostren una certa associació espacial entre cèl·lules activades per 20. L’activitat d’aquest factor sigma alternatiu és crucial per a la capacitat de recombinació de M. genitalium i per a la generació de variants genètiques dels antígens majoritaris d’aquest bacteri. En aquesta tesi, també ens hem centrat en l’estudi de la regulació de l’activitat de 20 per part de dues proteïnes no caracteritzades fins ara, anomenades RrlA i RrlB en aquest treball (recombination regulatory loci). Aquests reguladors es troben sota el control transcripcional de MG428 i estabilitzen la proteïna 20, permetent d’aquesta manera el progrés d’un cicle de retroalimentació positiva que és essencial per a la correcta activació del reguló de 20. Mutants tant de la proteïna RrlA com de la RrlB presenten deficiències de recombinació semblants al mutant defectiu de MG_428. Finalment, hem descrit una transferència horitzontal de DNA sense precedents entre soques de M. genitalium diferents. Aquesta transferència té lloc des d’una cèl·lula donadora que sobreexpressa 20 cap a una cèl·lula receptora mitjançant la recombinació homòloga.
En resum, en aquesta tesi doctoral hem presentat un nou regulador transcripcional de la recombinació a M. genitalium que promou la variació antigènica mitjançant l’activació de la transcripció d’un reguló únic. La proteïna 20 també permet l’activació d’un sistema de transferència horitzontal de gens poc ortodox que pot tenir un rol clau en l’evolució i adaptació d’aquests petits patògens.Mycoplasma genitalium is a small human pathogen that causes non-gonococcal urethritis, cervicitis and several pelvic inflammatory diseases. It bears one of the smallest genomes in an autonomous living organism and, for this reason, it has become a bacterial model for a minimal cell. Moreover, this small pathogen has a singular mechanism for antigenic variation that relies on the homologous recombination between the main adhesins and genomic repeats scattered around the chromosome. Despite that M. genitalium was one of the first microorganisms to be fully sequenced, very little is known of its reduced genetic circuitry and gene regulation. Besides, the regulatory mechanisms controlling the antigenic variation process are obscure notwithstanding it is essential for immune evasion and persistence. In this doctoral thesis, we have studied the role of the MG428 protein (20), which is an alternative sigma factor of this small bacterium. MG428 recognizes a novel promoter sequence and activates transcription of a small regulon composed by genes coding for key recombination enzymes, genes coding for hypothetical proteins and also non-coding regions with unknown function. Moreover, single cell analyses confirmed that activation via 20 only takes place in a small subset of the population at the same time and showed a certain spatial association between 20-activated cells. The activity of this alternative sigma factor is crucial for the recombination capacity displayed by M. genitalium and the generation of genetic variants of the main antigens in this bacterium. In this thesis, we have also focused on the regulation of 20 activity by two uncharacterized proteins, named RrlA and RrlB (recombination regulatory loci). These regulators are under the transcriptional control of MG428 and stabilize 20 protein, allowing the progression of a feed-forward loop required for the activation of the 20 regulon. Mutants of either rrlA or rrlB have similar recombination deficiencies as observed in an MG_428 null mutant. Finally, we have observed an unprecedented horizontal transfer of DNA between two different M. genitalium strains. DNA transfer occurs from a donor cell overexpressing 20 to a recipient cell by means of homologous recombination. Overall, in this doctoral thesis we have shown a novel transcriptional regulator of recombination in M. genitalium that promotes antigenic variation through the activation of transcription of a unique regulon. 20 can also activate an unorthodox horizontal gene transfer system that may play a key role in the evolution and adaptation of these small pathogens.
Book chapters on the topic "Antigen variation study"
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Bernander, Sverker, Berndt E. B. Claesson, Eva Hjelm, Nils Svensson, and Martin Hjorth. "Serologic Study of an Outbreak of Legionnaires' Disease: Variation of Sensitivity Associated with the Subgroup of Legionella pneumophila sg1 Antigen Used and Evidence of Concurrent Reactivity to Other Atypical Pneumonia Agents." In Legionella, 63–67. Washington, DC, USA: ASM Press, 2014. http://dx.doi.org/10.1128/9781555815660.ch17.
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Chappell, Lia, Sarah J. Lindsay, Phil Jones, Julian Parkhill, Jonathan Roberts, Nancy Holroyd, Michal Szpak, and Francesca Gale. "Parasite Genomics." In Genomics. Oxford University Press, 2020. http://dx.doi.org/10.1093/hesc/9780198848387.003.0005.
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This chapter focuses on parasite genomics. Parasitic infections may have beneficial effects on our immune systems, and the possibility that parasites have the potential to deliver previously unrecognized benefits as well as causing devastating harm makes it even more important to understand their biology. Some protozoan parasites, such as malaria and trypanosomes, use antigenic variation to evade destruction by the mammalian immune system. Genetic engineering can be used to manipulate the genomes of parasites and their vectors, and is a novel way to tackle disease transmission. Genomics can also give major insights into the mechanisms behind drug resistance in parasites. However, a limiting factor in parasite genomics is often the availability of parasite material: parasites can be small and live in places that are hard to access. Animal hosts and specialized environments are often needed to keep and study parasites in the lab.
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Erlich, Henry A., and Elizabeth A. Trachtenberg. "PCR-based methods of HLA typing." In Molecular Epidemiology, 181–208. Oxford University PressOxford, 2007. http://dx.doi.org/10.1093/oso/9780199638116.003.0007.
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Abstract Over the last two decades, molecular genetic techniques have been used to isolate the genes encoding the HLA class I and class II molecules and to characterize their genomic organization as well as their sequence diversity. The allelic sequence polymorphism at the HLA class I and II loci, revealed by extensive sequencing studies on a variety of human populations, is far greater than the antigenic variation detected by conventional serological typing (1). For example, there are about 280 DRB1 alleles defined by the second exon sequence but only about 15 different DR serological specificities or serotypes. Similarly, for the HLA-B locus, more than 450 alleles have been reported, compared to about 48 serological specificities. For the DPA1 and DPB1 loci, which encode the DP molecule, no serological typing system has been available and, consequently, most functional investigations of DP polymorphism, such as disease association studies or the analysis of DP mismatching in transplantation, have been possible only since the development of DNA-based HLA typing methods (2). The HLA class I and class II loci are the most polymorphic coding sequences in the human genome. The study of their allelic sequence diversity as well as the development of simple and rapid DNA-based typing methods has been greatly facilitated by the development of PCR amplification in the mid 1980s (3–6).
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"therefore be on the fourth SCR or on the serine/theonine rich region. By sequencing genomic DNA from Cr(a-) people, Telen and colleagues showed that a mutation in the fourth SCR was responsible for Cr3 [13]. Considering the MAIEA results, the fourth SCR would be a good place to start looking for difference responsible for the WES polymorphism too. Other Cromer system antigens showed some inhibition with one of the BRIC antibodies [12]. MAIEA provided biochemical evidence that Esa is indeed a Cromer system antigen [12]. Esa was thought to be a Cromer related antigen because of the failure of anti-Esa to react with Cromer-null cells and from its behaviour with proteinaese treated cells [14]. These findings were supported by the observation that Esa was carried by a glycosyl phosphatidylinositol linked protein [15]. However, only a small amount of anti-Esa was available and,therefore, immunoblotting experiments could not be done. Strong positive results with BRIC 216 and 110 but a negative result with BRIC 230 suggested that Esa is located on DAF, possibly on the first SCR. Similarly, a negative result with BRIC 230 and Tca suggests that it too is on the first SCR (Table II) [12]. The results of the MAIEA tests for Cromer antigens are summarised in Table II. They agree with those known from DNA studies, Dra on SCR III [15,16,17] and Cr3 on SCR IV [13], and suggest the best places to look for those as yet undetermined. This demonstrates how MAIEA may be used to help narrow the field of study to determine the molecular basis of antigens. VARIATION IN EXPRESSION OF SOME Rh ANTIGENS We had hoped to apply MAIEA to Rh but to date the only antibodies to the D protein are of human origin, so MAIEA cannot yet be used to study the relationship of the D antigen to some of the low incidence antigens which appear to be markers of partial D antigens. The Rh antigen D is, after ABO, the most important antigen clinically because it is highly immunogenic. Until the introduction of Rh immunoprophylaxis, anti-D was the most frequent cause of haemolytic disease of the newborn and neonatal death [1]. Many Rh antigens are good immunogens. Since its initial recognition in the nineteen-forties, the Rh system has become very complex. There are 48 numbered antigens, that is serologically defined determinants, the numbers have reached 50 because two numbers have been declared obsolete [2,3,18,19]. Some antigens are polymorphic and others are of high or low incidence." In Transfusion Immunology and Medicine, 191. CRC Press, 1995. http://dx.doi.org/10.1201/9781482273441-10.
Full textConference papers on the topic "Antigen variation study"
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Grimaudo, V., E. K. O. Kruithof, J. Hauert, and F. Bachmann. "DIURNAL VARIATION OF COMPONENTS OF THE FIBRINOLYTIC SYSTEM." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1644381.
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To elucidate the causes of the well known diurnal variations of the fibrinolytic activity we have studied severed parameters of the fibrinolytic system in 8 healthy male volunteers during a period of 24 h. Blood sanples were taken at 8, 10 and 12 a.m., 4 and 8 p.m. and at 8 a.m. next morning. Hie following parameters were analyzed: euglobulin clot lysis time (ECLT), fibrinolytic activity of euglobulins on fibrin plates (FPA), antigen concentrations of tissue-type plasminogen activator (t-PA) by ELISA and of urokinase (u-PA) and plasminogen activator inhibitor-1 (PAI-1) by RIA, the overall PA-inhibitor activity by the indirect chranogenic substrate assay (Verheijen et al; 1984) and the electrophoretic-zymogra-phic analysis of the euglobulins fraction.Global fibrinolytic activity increased from 1.0 ±0.3 U/ml at 8 a.m. to 2.5 ±1.1 U/ml at 8 p.m. (p <0.002) as measured by ECLT (U=300/lysis time in minutes) and from 2.3 ±0.4 IU/ml to 4.7 ±1.1 IU/ml (p <0.001) as determined by FPA. In contrast, t-PA antigen decreased from 5.1 ±0.8 ng/ml at 8 a.m. to 2.9 ±1.5 ng/ml at 8 p.m. (p<0.01) and u-PA antigen from 8.1 ±1.2 ng/ml to 7.1 ±0.7 ng/ml (p<0.05). Most pronounced was the decrease of PAI-1 antigen from 23.5 ± 7.6 ng/ml in the morning to 8.8 ±2.6 ng/ml at 8 p.m. (p <0.001), while PAI activity decreased from 8.8 ±3.6 U/ml to 5.5 ±1.8 U/ml at 4 p.m. (p <0.002). During the entire study period the electro-phoretic-zymographic analysis did not reveal any free t-PA; the latter was present throughout in the form of the 110 kD t-PA/PAI-1 ccnplex. During the day the intensity of the 110 kD band decreased progressively.These findings confirm the physiological diurnal fluctuation of the fibrinolytic activity. All components of the fibrinolytic system are involved in this fluctuation and participate in the modulation of fibrinolytic activity.Our study demonstrates that the diurnal increase of fibrinolytic activity is due principally to a marked decline of the PAI-1 concentration during the day and not to an increase of PA antigen levels.
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Gunda, Naga Siva Kumar, and Sushanta K. Mitra. "Microfluidic Based Biosensor for Detection of Cardiac Markers." In ASME 2013 Fluids Engineering Division Summer Meeting. American Society of Mechanical Engineers, 2013. http://dx.doi.org/10.1115/fedsm2013-16270.
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Myocardial Infarction (MI) occurs when the blood flow to the heart is blocked. It is major threat to human kind. Current laboratory and ELISA tests are expensive, time consuming, and are not very sensitive. Biosensors can play an important role in the diagnosis of MI without relying on hospital visits. Therefore, researchers are focusing to develop rapid, hand-held, inexpensive biosensors for detecting cardiac markers. In the present study, one of the cardiac markers (Troponin T) is detected using microfluidic based biosensor. Troponin T (cTnT) releases in to the blood serum within 4–6 h after minor heart attack and remains elevated for up to 2 weeks, which will help in diagnosing the heart condition. In this work, a microfluidic channel with an array of gold strips is considered for detecting and quantifying the Troponin T in an aqueous solution. Troponin T primary (capture) antibody is immobilized on gold strip using self assembled monolayer (SAM) consisting of a homogeneous mixture of oligo (ethylene glycol) (OEG)-terminated alkanethiolate and mercaptohexadecanoic acid (MHDA). Then, an aqueous solution containing Troponin T antigen is injected into the microchannel to facilitate antibody-antigen reaction to take place in less time. Later, FITC tagged Troponin T secondary (detection) antibody is dispensed in to the channel for quantification of Troponin T antigen. Using confocal fluorescent reader, the variation of fluorescent intensity across the microchannel is measured and quantified the concentration of Troponin T antigen with calibrated samples. Contact angle measurement system, Fourier Transform Infrared Spectroscopy and Ellipsometer are used to characterize the surface properties at each stage of biomolecule immobilization.
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Mathew, Shilu M., Malak Ibrahim, Asmaa Al Thani, Khalid Al Ansari, Hassan Zaraket, and Hadi M. Yassine. "Antigenica and Genetic Characterization of Identified Rotavirus Strains in Qatar in Response to Rotarix Vaccine Usage." In Qatar University Annual Research Forum & Exhibition. Qatar University Press, 2020. http://dx.doi.org/10.29117/quarfe.2020.0114.
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To identify genetic and antigenic variation in RV in response to vaccine usage. Methods: A total of 231 RV-positive fecal samples were collected from children suffering from AGE during three-year study period between June 2016 and June 2019. The age of the subjects ranged between 2 months and 14 years (median of 16 months). RV genotyping and neutralizing regions, which include both VP4 (Ptype) and VP7 (G type), were amplified and sequenced. We characterized amino acid sequence variability and predicted antigenicity compared to the Rotarix vaccine strain. Phylogenetic analyses were performed using MEGA7.0. Fisher’s exact test was used to run the statistical analysis for the clinical and demographical characteristics of circulating strains. Results: RV infection was most common in children between 3-36 months of age. Among the RV-positive cases, 135 (59.3%) had been vaccinated using either of the RV vaccines available. The number of children vaccinated with one and two-dose was 53 (39.2%) and 82 (60.8%), respectively. The percentage reduction of disease in a vaccinated group of pediatrics compared to an unvaccinated group of pediatrics was 25%. Of these, 108 (78.2%) experienced diarrhea for less than three days, and only eight (6.7%) had diarrhea for more than five days. All vaccinated children showed mild to moderate dehydration except for ten children who were then treated with intravenous fluids. G3 strains were the most strains detected (40%) followed by G2 (17.7%), G4 (16.8%), G9 (15%), G1 (9%), and G8 (0.9%). The dominant RV strains during the study period were G3P [8] (30.8%), G2P [8] (12.3%), G4P [8] (11.7%) and G1P[8] (10.4%). Comparisons of the amino acid residues defining the VP7 and VP4 antigenic domains revealed several mismatches between G1P [8] strains and the G1 and P [8] strains contained in the currently licensed rotavirus vaccines Rotarix. Eighty percent (n=8) of the G1 genotype specimens harbored three amino acid substitutions (N94S, S123N, and M217T) in 7‐ 1a and 7‐ 2b antigenic sites in comparison to the Rotarix vaccine. The P [8] strains with G4 and G9 counterparts showed the highest degree of variation among all specimens with known G genotype. These viruses had 15 and 13 substitutions in their VP4 antigenic epitopes when compared with the P [8] component of the Rotarix vaccines. Conclusion: This study suggests genetic variability in G1 genotype specimens to escape the vaccine-derived immune response. It also identified the wide diversity of circulating RV genotypes in Qatar.
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Celik, Emrah, Nicolas Rongione, Amelia Bahamonde, Zheng Ao, and Ram Datar. "Isolation of Circulating Tumor Cells Using Stiffness-Based Filtration Platform." In ASME 2015 International Mechanical Engineering Congress and Exposition. American Society of Mechanical Engineers, 2015. http://dx.doi.org/10.1115/imece2015-53241.
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Analysis of isolated cancer cells in circulation is proven to help determine the success of the cancer treatment and understand the genetic signature of cancer disease. Scarcity of these cells in blood circulation (1–10 CTC in 1ml blood) however, makes the isolation process extremely challenging. Ever improving CTC isolation methods fall into two main categories: 1.Immunomagnetic separation based on antibody binding to tumor specific biomarkers expressed on the cell 2. Physical separation based on the size of the CTCs. Efficiency in cell isolation is still low in these techniques due to the variation in expression level of tumor specific antigens and tumor cell size. Therefore, tumor cell isolation strategies using new CTC biomarkers must be explored. In this study, we investigated the feasibility of using mechanical stiffness difference in order to detect and isolate the circulating tumor cells from the blood cells. AFM nanindentation experiments revealed that cancer cells are significantly softer than the surrounding white blood cells and therefore, stiffness can be used as a biomarker for CTC isolation. In addition, finite element analysis simulations have shown that CTC isolation can be performed at high efficiency using stiffness-based isolation. Therefore, stiffness based isolation has a potential to achieve fast, label-free isolation of CTCs at high efficiency for clinical applications.
Reports on the topic "Antigen variation study"
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Yogev, David, Ricardo Rosenbusch, Sharon Levisohn, and Eitan Rapoport. Molecular Pathogenesis of Mycoplasma bovis and Mycoplasma agalactiae and its Application in Diagnosis and Control. United States Department of Agriculture, April 2000. http://dx.doi.org/10.32747/2000.7573073.bard.
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Mycoplasma bovis and M. agalactiae are two phylogenetically related mycoplasmas which cause economically significant diseases in their respective bovine or small ruminant hosts. These organisms cause persistent asymptomatic infections that can result in severe outbreaks upon introduction of carrier animals into susceptible herds. Little is known about the mechanisms underlying mycoplasma-host interaction, variation in virulence, or of the factors enabling avoidance of the host immune system. In recent years it has become apparent that the ability of pathogenic microorganisms to rapidly alter surface antigenic structures and to fine tune their antigenicity, a phenomena called antigenic variation, is one of the most effective strategies used to escape immune destruction and to establish chronic infections. Our discovery of a novel genetic system, mediating antigenic variation in M. bovis (vsp) as well as in M. agalactiae (avg) served as a starting point for our proposal which included the following objectives: (i) Molecular and functional characterization of the variable surface lipoproteins (Vsp) system of M. bovis and comparison with the Vsp-counterpart in M. agalactiae (ii) Determination of the role of Vsp proteins in the survival of M. bovis when confronted by host defense factors, (iii) Assessment of Vsp-based genetic and antigenic typing of M. bovis and M. agalactiae for epidemiology of infection and (iv) Improvement of diagnostic tests for M. bovis and M. agalactiae based on the vsp-and vsp-analogous systems. We have carried out an extensive molecular characterization of the vsp system and unravelled the precise molecular mechanism responsible for the generation of surface antigenic variation in M. bovis. Our data clearly demonstrated that the two pathogenic mycoplasma species possess large gene families encoding variable lipoprotein antigens that apparently play an important role in immune evasion and in pathogen-host interaction during infection. Phase variable production of these antigens was found to be mediated by a novel molecular mechanism utilizing double site-specific DNA inversions via an intermediate vsp configuration. Studies in model systems indicate that phase variation of VspA is relevant in interaction between M. bovis and macrophages or monocytes, a crucial stage in pathogenesis. Using an ELISA test with captured VspA as an antigen, phase variation was shown to occur in vivo and under field conditions. Genomic rearrangements in the avg gene family of M. agalactiae were shown to occur in vivo and may well have a role in evasion of host defences and establishment of chronic infection. An epidemiological study indicated that patterns of vsp-related antigenic variation diverge rapidly in an M. bovis infected herd. Marked divergence was also found with avg-based genomic typing of M. agalactiae in chronically infected sheep. However, avg-genomic fingerprints were found to be relatively homogeneous in different animals during acute stages of an outbreak of Contagious Agalactiae, and differ between unrelated outbreaks. These data support the concept of vsp-based genomic typing but indicate the necessity for further refinement of the methodology. The molecular knowledge on these surface antigens and their encoding genes provides the basis for generating specific recombinant tools and serological methods for serodiagnosis and epidemiological purposes. Utilization of these methods in the field may allow differentiating acutely infected herds from chronic herds and disease-free herds. In addition the highly immunogenic nature of these lipoproteins may facilitate the design of protective vaccine against mycoplasma infections.
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Levisohn, Sharon, Mark Jackwood, and Stanley Kleven. New Approaches for Detection of Mycoplasma iowae Infection in Turkeys. United States Department of Agriculture, February 1995. http://dx.doi.org/10.32747/1995.7612834.bard.
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Mycoplasma iowae (Mi) is a pathogenic avian mycoplasma which causes mortality in turkey embryos and as such has clinical and economic significance for the turkey breeder industry. Control of Mi infection is severely hampered by lack of adequate diagnostic tests, together with resistance to most antibiotics and resilience to environment. A markedly high degree of intra-species antigenic variation also contributes to difficulties in detection and control of infection. In this project we have designed an innovative gene-based diagnostic test based on specific amplification of the 16S rRNA gene of Mi. This reaction, designed Multi-species PCR-RFLP test, also amplifies the DNA of the pathogenic avian mycoplasmas M. gallisepticum (Mg) and M. synoviae (Ms). This test detects DNA equivalent to about 300 cfu Mi or either of the other two target mycoplasmas, individually or in mixed infection. It is a quick test, applicable to a wide variety of clinical samples, such as allantoic fluid or tracheal or cloacal swab suspensions. Differential diagnosis is carried out by gel electro-phoresis of the PCR amplicon digested with selected restriction enzymes (Restriction Fragment Length Polymorphism). This can also be readily accomplished by using a simple Dot-Blot hybridization assay with digoxigenin-labeled oligonucleotide probes reacting specifically with unique Mi, Mg or Ms sequences in the PCR amplicon. The PCR/OLIGO test increased sensitivity by at least 10-fold with a capacity for rapid testing of large numbers of samples. Experimental infection trials were carried out to evaluate the diagnostic tools and to study pathogenesis of Mi infection. Field studies and experimental infection of embryonated eggs indicated both synergistic and competitive interaction of mycoplasma pathogens in mixed infection. The value of the PCR diagnostic tests for following the time course of egg transmission was shown. A workable serological test (Dot Immunobinding Assay) was also developed but there was no clear-cut evidence that infected turkeys develop an immune response. Typing of a wide spectrum of Mi field isolates by a variety of gene-based molecular techniques indicated a higher degree of genetic homogeneity than predicted on the basis of the phenotypic variability. All known strains of Mi were detected by the method developed. Together with an M. meleagridis-PCR test based on the same gene, the Multi-species PCR test is a highly valuable tool for diagnosis of pathogenic mycoplasmas in single or mixed infection. The further application of this rapid and specific test as a part of Mi and overall mycoplasma control programs will be dependent on developments in the turkey industry.