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Coccidioidomicose no estado de Cearà (1995-2007): caracterÃsticas clÃnico-laboratoriais e anÃlise das fraÃÃes protÃicas do antÃgeno total de Coccidioides posadasii no imunodiagnÃstico / Coccidioidomycosis State of Cearà (1995 - 2007): characteristics clinical laboratory and analysis of protein fractions of antigen total Coccidioides posadasii in the immunodiagnosisSilviane Praciano Bandeira 04 December 2008 (has links)
Coccidioidomicose consiste em enfermidade causada pelos fungos dimÃrficos do gÃnero Coccidioides â C. immitis e C. posadasii â que acomete o homem e diversos animais como bovinos, ovinos, roedores e cÃes. Restrita ao continente americano, casos da doenÃa vem ocorrendo no Estado do Cearà desde 1995. A infecÃÃo ocorre geralmente por contato inalatÃrio com as estruturas infectantes do fungo presentes no ambiente, os artroconÃdeos. Em sua fase parasitÃria, os microrganismos se apresentam sob a forma de esfÃrulas responsÃveis pelas manifestaÃÃes clÃnicas da doenÃa. A
maioria dos casos cursa de forma assintomÃtica, sendo os quadros respiratÃrios os mais comuns em pacientes clinicamente enfermos. O diagnÃstico da doenÃa se baseia
em tÃcnicas microbiolÃgicas como pesquisa direta e cultura, porÃm estes procedimentos demandam estrutura especializada â nÃvel de biosseguranÃa III -por tratar-se de microrganismo de classe biolÃgica 3. Abordagem imunolÃgica mostra-se promissora por prescindir da manipulaÃÃo fÃngica e ser exeqÃÃvel em diversos laboratÃrios de rotina micolÃgica. Neste trabalho, objetivou-se traÃar perfil clÃnicolaboratorial
dos casos de coccidioidomicose ocorridos no Estado do Cearà entre os anos de 1995 e 2007, alÃm de obter fraÃÃes antigÃnicas a partir do fracionamento de AntÃgeno total de C. posadasii empregÃveis como imunodiagnÃstico da doenÃa. Foram catalogados 19 casos da doenÃa no perÃodo em estudo. Todos os pacientes eram homens jovens com manifestaÃÃes respiratÃrias, com exceÃÃo de um caso. O hÃbito recreativo de caÃar tatus foi relatado por 18 pacientes. A abordagem laboratorial destes casos consistiu em estudo microbiolÃgico, associado a tÃcnicas moleculares, imunolÃgicas, histopatolÃgicas ou de experimentaÃÃo animal. O fracionamento protÃico do AntÃgeno total de C. posadasii originou trÃs fraÃÃes antigÃnicas com teor protÃico distinto, perfil eletroforÃtico diferenciado e imunorreatividade pela tÃcnica de Western blotting. A fraÃÃo 60-90% revelou banda imunorreagente de aproximadamente 30 a 40 KDa reconhecida por soros de coccidioidomicose, porÃm apresentando reatividade cruzada com alguns soros de paciente com histoplasmose.
Esta banda protÃica, em estudos posteriores, poderà ser caracterizada e purificada com potencial utilizaÃÃo como ferramenta para o imunodiagnÃstico da doenÃa. / Coccidiodomycosis is a disease caused by dimorphic fungi of the Coccidioides genus â C. immitis and C. posadasii â which afflicts people and various animals, such as cattle, goats, rodents and dogs. It is restricted to the Americas and cases have been reported in the state of Cearà only since 1995. The infection generally results from inhaling the infectious structures arthroconidia -of the fungus. In its parasitic phase, the microorganisms responsible for the clinical signs of the disease have a spherical shape. Most cases are asymptomatic, and respiratory problems are the most common symptoms in patients that are clinically affected. The diseaseâs diagnosis is based on microbiological techniques such as direct examination of
clinical specimens and culturing, but these procedures require specialized biosecurity level 3 facilities because the microorganism falls in biological class 3. The immunological approach appears promising because it does not require manipulation of the fungi and can be performed
in various laboratories equipped for routine mycological tests. The aim of this work is to outline the clinical and laboratory profile of the cases of coccidiodomycosis that occurred in the state of Cearà between 1995 and 2007 and to report experiments to obtain antigenic fractions from fractioning AntÃgeno total of C. posadasii, as a possible means for immunodiagnosis of the disease. A total of 19 cases of the disease were cataloged in the study period. All the patients were young men with respiratory symptoms, except for one case. All but one of the patients reported they engaged in recreational hunting of armadillos. The
laboratory approach in these cases consisted of microbiological examination combined with molecular, immunological, histopathological techniques or animal experiments. The protein fractioning of AntÃgeno total of C. posadasii produced three antigenic fractions with different protein content, electrophoretic profile and immunoreactivity according to the Western blotting technique. The 60-90% fraction showed an immunoreactive band of approximately 30 to 40 KDa, recognizable by sera from patients with coccidiodomycosis, but showed crossreactivity with some sera from patient with histoplasmosis. This protein band, in subsequent
studies, can be characterized and purified for potential use as a tool for immunodiagnosis of
the disease.
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Partial characterization and purification of a murine suppressor-inducer factor secreted by a natural supressor cell line and characterizaton of the binding of antisera raised to murine antigen-specfic suppressive material in human peripheral leukocytesNorman, Nadine Elizabeth January 1990 (has links)
A large amount of effort has gone into the elucidation of the mechanism of suppression of the immune system. This level of immunoregulation has been demonstrated to be mediated by both antigen-nonspecific and antigen-specific protein factors elicited by leukocytes.
In this work, two different modes of immunosuppression were investigated. First, an attempt was made to purifiy an antigen-nonspecific protein factor, SIF (Suppressor Inducer Factor) secreted by the Natural Suppressor cell line M1-A5. M1-A5 culture supernatants were subjected to ion exchange chromatogrphy (IEC) and fast protein liquid chromatography (FPLC). Bioactivity of eluted fractions was determined by the plaque forming cell assay and followed through the purification. Reducing SDS-PAGE of selected fractions suggested that bands with Mr's of >110 KD and/or 55 KD were mediating the suppressive activity. In addition, an assay was developed to further investigate the mode of action of SIF.
Second, the binding of two antisera raised to components associated with murine antigen-specific suppression was studied using human peripheral leucocytes and several human tumour cell lines. Anti-p80 and anti-p30 binding was found to be variable (within a range) and to involve two populations of human mononuclear cells. Subsequently, it was found that all CD3⁺ (T cells), CD19⁺ (B cells) and neutophils expressed both the p80 and p30 determinants.
Four human leukemic cell lines were found to express varying levels of the p80 and p30 determinants. Cell lysates from each of the cell lines were subjected to Western blot analyses using anti-p30. The results showed that anti-p30 binds to a major band of 42 KD and minor bands of 60 and 80 KD in all lysates. In addition, a 25 KD band was observed in RAJI and CEM-CM3 lysates only. Thus, it appears that HuT 78 cells sythesize but are unable to express the p30-containing, 42 KD molecule on the cell surface.
No firm conclusions can be made with respect to the biochemical nature or mechanisism of action of either of the two suppressor factors studied in this work. Research into the mechanisms of suppression of the immune system is complex and multi-faceted, and it seems that for now, there will remain a gap in our overall understanding of immune regulation. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate
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Purification, biochemical analysis and sequencing of a novel murine T suppressor factorChan, Agnes How-Ching January 1988 (has links)
The work reported in this thesis involved the purification, biochemical analysis and sequencing of a novel suppressor factor secreted by a T cell hybridoma, A10. The factor, A10F, isolated from spent medium of A10 cells, was found to consist of two forms with molecular weights at 140 - 160 and 80 kD as suggested by NH₂-terminal sequencing, Western blotting and tryptic peptide mapping experiments. Both forms of A10F were found to be capable of suppressing the in vitro generation of cytotoxic T lymphocyte (CTL) specific for P815 cells by syngeneic (DBA/2) splenocytes.
In vitro ³⁵S methionine labeling experiments clearly demonstrated that the 80 kD protein was a secretory product of the A10 cells. The protein, which was specific to the monoclonal antibody (B16G), was absent in the control NS1 and BW5147 cells. Biochemical analysis indicated that the 80 kD molecule, was either a degradation product or a monomer of the 140 - 160 kD molecule. Further degradation products such as the 32 kD molecules were also found. This peptide, however, did not seem to cause substantial suppression in the in vitro CTL assay.
When the 140 - 160, 80 and 32 kD proteins were sequenced at the NH₂ terminus, both 140 - 160 and 80 kD proteins were found to possess the same NH₂ terminus sequence. The 32 kD protein, on the other hand, was found to have an
NH₂-terminus quite different from that of the 80 kD protein. These findings suggested that the 32 kD fragment was probably located at the distal end of the 140 - 160 kD molecule. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate
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Immunological techniques in the investigation of the physiological functions of gastric inhibitory polypeptide and motilinDryburgh, Jill Robertson January 1977 (has links)
A radioimmunoassay was developed, specific for the gastrointestinal polypeptide, motilin. Antisera were raised in guinea pigs and rabbits. The immunogen was porcine motilin, conjugated to bovine serum albumin by the carbodiimide condensation
reaction. The routine antiserum behaved identically towards endogenously-
released motilin and the pure standard preparation. A radioactive tracer of high
125
specific activity was obtained after incorporation of - iodine into the motilin molecule by the chloramine-T method. The optimum conditions for all other assay variables were established to produce the most sensitive displacement Cstandard) curve. Motilin antiserum, coupled directly to an agarose matrix, retained full antibody activity and sensitivity. It is a feasible technique for use in both the radioimmunoassay and in the extraction of motilin from both serum and tissue extracts.
The fasting serum levels of IR- motilin was 190 - 131 pg/ml in men and 294 -'.44 pg/ml in dogs (mean - SD) . The increase in motor activity in the extrinsically denervated fundic pouch of the dog after duodenal alkalinization was associated with a concomitant elevation in serum IR- motilin levels. This increase in serum IR- motilin was in the same range as that achieved by the exogenous administration of the porcine polypeptide which produced the same motor response. Duodenal acidification produced an apparent increase in serum IR-motilin with no associated increase in gastric motor activity. Only one peak of motilin immunoreactivity was detected when serum containing alkali-stimulated motilin or a partially purified duodenal extract were subjected to gel filtration on Sephadex G-50. The distribution of motilin throughout the hog gastrointestinal tract, determined by radioimmunoassay on partially purified extracts, agreed with
the immunocytochemical findings that motilin was predominantly located in the duodeninn and jejunum, with traces.in the upper ileum.
Virtually the intact molecule was required for the expression of full biological
potency. The individual amino acids were important inasmuch as they contributed to the charge distribution and conformation of the molecule.
The physiological release and function of motilin have yet to be determined. Elevated levels of circulating IR- motilin have not been associated with any gastro-intestinal function, although they appear to be depressed by feeding. Motilin has been implicated in the control of the interdigestive phase of gastric motor activity. It may be acting in a local or paracrine manner. Motilin has not been implicated in any .•cU'in±cal.rst"ait"eC&s sjffetfce i
The hormonal status of gastric inhibitory polypeptide (GIP) has been studied with the existing radioimmunoassay, modified to improve the label specific activity (by ion exchange chromatography). Direct coupling of GIP antisera to agarose beads was unsatisfactory, antibody activity and sensitivity being greatly reduced by the close proximity of the solid matrix. The postulated role of GIP as the enterogastrone1 of Kosaka and Lim, suggested by studies with exogenously-administered polypeptide, was confirmed by experiments in the dog. Pentagastrin-stimulated gastric acid secretion was inhibited by intra-duodenal infusion with glucose or fat; this inhibition being associated with a significant
elevation in the circulating serum IR- GIP levels, within the range produced
by ingestion of a mixed meal. GIP does not appear to be involved in the inhibition of gastric acid secretion produced by duodenal acidification.
Endogenous;GIP.stimulated by either fat or glucose exhibited at least 3 Immunoreactive components after column chromatography. The IR- GIP eluting in the void volume appeared to represent a non-specific complex between GIP and a serum protein and is possibly biologically inactive. A second IR-GIP component with a molecular weight of 7500-8000 (ProGIP), eluted ahead of the established form of GIP (molecular weight = 5105). ProGIP has been found to be relatively unstable. ProGIP and GIP^QQQ have also been detected in extracts of hog duodenal mucosa. The established insulinotropic effect of GIP correlates best with that percentage of the total IR- GIP composed of ProGIP and GIP500(). The relative proportions of IR- GIP500Q and IR- ProGIP in serum samples taken at different times after ingestion of either fat or glucose, suggest that ProGIP is either a precursor of GIP or that the ProGIP-producing cells occupy a more distal region of the duodenal and jejunal mucosa than the GIP- producing cells.
Exogenous administration of synthetic somatostatin in dogs and man will inhibit both.GIP release by either fat or glucose and the insulino-tropic action of GIP at the level of the 8/-cell. Naturally-occurring intestinal or pancreatic somatostatin may contribute to the control of GIP release and serve to modulate the GIP- mediated response of the gastric parietal or pancreatic β-cell. / Medicine, Faculty of / Cellular and Physiological Sciences, Department of / Graduate
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Evaluation of a recombinant rift valley fever virus nucleocapsid protein as a vaccine and an immunodiagnostic reagentVan Vuren, Petrus Jansen 17 January 2012 (has links)
The serodiagnosis of Rift Valley fever (RVF) relies on the use of inactivated whole virus based reagents
which present biosafety, financial and operational constraints. There are no vaccines for humans, the
availability of animal vaccines is limited and they have several drawbacks. The aim of this study was to
evaluate a bacterially expressed recombinant RVF virus (RVFV) nucleocapsid protein (recNP) as a safe
immunodiagnostic reagent, and an immunogen in a mouse and host animal model. Several enzyme-linked
immunosorbent assays (ELISAs) were developed in this study, enabling sensitive and specific detection
of antibodies and RVFV antigen in human and animal specimens. The recNP was combined with
different adjuvants and used to immunize mice and sheep subsequently challenged with a virulent wild
type RVFV strain. Depending on the recNP/adjuvant combination, protection against disease in mice
ranged between 17 and 100%, with sterilizing immunity elicited in some experimental groups, compared
to 100% morbidity/mortality and excessive viral replication in adjuvant and PBS control mice.
Immunization with recNP combined with Alhydrogel, an adjuvant that biases immunity towards Th2
humoral immunity, that yielded 100% protection, induced an earlier and stronger type I interferon
response in mice after challenge, compared to repression of the same gene in adjuvant and PBS control
mice. There was massive activation of pro-inflammatory responses and genes with pro-apoptotic effects
in the livers of control mice at the acute phase of infection, accompanied by high viral replication,
possibly contributing to the pathology of the liver. There was also evidence of activation and repression
of several genes involved in activation of B- and T-cell immunity in control mice, some indicating
possible immune evasion by the challenge virus. Immunization of sheep with the same recNP/adjuvant
combinations were, however, not able to decrease replication of challenge virus. The recNP based
ELISAs are an important addition to and improvement of the currently available serodiagnostic tests for
RVF. The mechanism by which recNP immunization protects mice from developing severe disease
during the acute phase of infection is now better understood, but the mechanism for earlier clearance of
the virus needs further investigation.
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TRAPC : a novel triggering receptor expressed on antigen presenting cells /Holst, Rutger van der, January 2007 (has links)
Diss. Stockholm : Karolinska institutet, 2007.
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Models for infections in immunodeficiency /Berglöf, Anna, January 2002 (has links)
Diss. (sammanfattning) Stockholm : Karol. inst., 2002. / Härtill 5 uppsatser.
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Modulation of natural killer cell and T-cell functions by CD94/NKG2A receptors /Teixeira de Matos, Cristina, January 2006 (has links)
Diss. (sammanfattning) Stockholm : Karolinska institutet, 2006. / Härtill 4 uppsatser.
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Regulation of Fas ligand (CD178) in murine CD8+ cytotoxic T lymphocyte populationsMartin, James Sean. January 2008 (has links)
Thesis (Ph. D.)--University of Virginia, 2008. / Title from title page. Includes bibliographical references. Also available online through Digital Dissertations.
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The host immune response to Streptococcus pneumoniae : bridging innate and adaptive immunity /Lee, Katherine Shi-Hui January 2006 (has links) (PDF)
Thesis (Ph.D.)--Uniformed Services University of the Health Sciences, 2006 / Typescript (photocopy)
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