No description available.

Diagnostic medical mycology has been slow to advance due to a lack of species specific antigens in organisms which cause serious diseases in man. Toward this end, an HPLC analysis was done of the following fungal antigens: histoplasmins HKC-43 and H-42, blastomycin KCB-26, an alkali-soluble, water soluble antigen from Blastomyces dermatitidis (b-ASWS), a coccidioidin prepared from a toluene lysate of the mycelial-arthroconidia phase of Coccidioides immitis, and an alkali-soluble, water-soluble antigen from Coccidioides immitis (c-ASWS). The HPLC survey included size-exclusion chromatography (SEC), ion exchange chromatography (HPIEC), and reversephase chromatography (RP). Resolution was poor with both SEC and HPIEC but was excellent with RP chromatography. The use of RP will allow sufficient separation for further antigenic and structural analysis.

abstract: Valley Fever (VF), is a potentially lethal fungal pneumonia caused by Coccidioides spp., which is estimated to cause ~15-30% of all community-acquired pneumonias in the highly endemic Greater Phoenix and Tucson areas of Arizona. However, an accurate antigen-based diagnostic is still lacking. In order to identify protein and glycan antigen biomarkers of infection, I used a combination of genomics, proteomics and glycomics analyses to provide evidence of genus-specific proteins and glycosylations. The next goal was to determine if Coccidioides-specific glycans were present in biological samples from VF patients. Urine collected from 77 humans and 63 dogs were enriched for glycans and evaluated by mass spectrometry for Coccidioides-specific glycans and evaluated against a panel of normal donor urines, urines from patients infected with other fungi, and fungal cultures from closely related pneumonia-causing fungi. A combination of 6 glycan biomarkers was 100% sensitive and 100% specific in the diagnosis of human VF subjects, while only 3 glycan biomarkers were needed for 100% sensitivity and 100 specificity in the diagnosis of dog VF subject. Additionally, a blinded trial of 23 human urine samples was correctly able to classify urine samples with 93.3% sensitivity and 100% specificity. The results of this research provides evidence that Coccidioides genus-specific glycosylations have potential as antigens in diagnostic assays. / Dissertation/Thesis / Doctoral Dissertation Microbiology 2019

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / FundaÃÃo de Amparo a CiÃncia e Tecnologia / Coccidioidomicose à uma micose sistÃmica que acomete o homem e outros animais domÃsticos e silvestres, causada pelo fungo dimÃrfico geofÃlico Coccidioides posadasii. Nos Ãltimos anos, a melhoria dos mÃtodos de diagnÃstico micolÃgico e o aumento da ocorrÃncia de doenÃas imunossupressoras causaram grande impacto na incidÃncia das micoses profundas e oportunistas no mundo. Apesar da existÃncia de terapias antifÃngicas eficazes contra a coccidioidomicose, a busca por novas drogas para o tratamento desta doenÃa se faz necessÃria. O objetivo desse estudo foi determinar o efeito inibitÃrio, in vitro, e o mecanismo de aÃÃo dos anÃlogos quÃmicos da isoniazida, isolados e em combinaÃÃo com os antifÃngicos anfotericina B (AMB) e itraconazol (ITC), frente a cepas de Coccidioides posadasii (n=12). Para isso 12 cepas de C.posadasii, foram repicadas em Ãgar dextrose batata e utilizadas na realizaÃÃo dos testes de sensibilidade frente ao agente antituberculose isoniazida (INH) e seus anÃlogos quÃmicos. Os ensaios foram conduzidos por meio do teste de macrodiluiÃÃo em caldo ou definiÃÃo da concentraÃÃo inibitÃria mÃnima (CIM) e o sinergismo foi avaliado pelo mÃtodo do tabuleiro. Os anÃlogos quÃmicos inibiram o crescimento de todas as cepas de C. posadasii testadas isoladamente, com valores de CIM para cada composto que variaram de 100 a 400 Âg/mL, para os anÃlogos P3-[Nâ-(1- (4-methoxifeniletilideno) isonicotinohidrazida] P5- [Nâ- (1-m-tolileilidene) isonicotinohidrazida] e P6- [Nâ-(1-(4-metilfenil) etilideno) isonicotinohidrazida], de 25 a 100 Âg/mL para a PACT- Nâ- (1-feniletilideno) isonicotinohidrazida e de 0,0625 a 0,125 Âg/mL para AMB e 0,125 a 0,5 Âg/mL para ITC. Quanto a combinaÃÃo entre as drogas antifÃngicas e os anÃlogos quÃmicos da droga isoniazida, todos foram capazes de inibir o crescimento in vitro das cepas de C. posadasii. Sinergismo foi detectado em 8 combinaÃÃes nÃo sendo observado nenhum antagonismo em nenhuma delas. Os anÃlogos da isoniazida apresentaram valores de CIM de 2, 4, 8, 16, 32 vezes superior à atividade da droga antituberculose padrÃo. A extraÃÃo dos esterÃis e a permeabilidade da membrana fÃngica foram averiguadas, respectivamente, por saponificaÃÃo com aquecimento e por meio da leitura do sobrenadante da suspensÃo fÃngica em comprimento de onda de 260 e 280 nm. Todos os compostos inibiram o crescimento in vitro das cepas de C. posadasii. Foi observado que os anÃlogos da isoniazida isolados e em combinaÃÃo com antifÃngicos foram capazes de causar reduÃÃo do teor de ergosterol celular; apenas os anÃlogos P3 e P6 foram capazes de alterar a permeabilidade da membrana plasmÃtica nas condiÃÃes testadas. Em conclusÃo, os resultados mostram que os anÃlogos derivados da isoniazida possuem atividade inibitÃria frente a C. posadasii

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / Coccidiodomycosis is a systemic infection, predominantly pulmonary, caused by the geophilic and dimorphic fungi, Coccidioides immitis and Coccidioides posadasii. In Brazil, coccidioidomycosis is associated with semi-arid areas in the Northeastern region of this country, which is considered one of the endemic areas of this disease in South America. These pathogens are morphologically indistinguishable species, but they exhibit molecular differences. Different molecular techniques have been described for the characterization of these species. The nuclear ribosomal DNA (rDNA) from Coccidioides spp. has been described as an important molecular marker for the identification, taxonomy and phylogeny. Currently, there are still shortages of maps for epidemiological approaches in order to direct the correlation between populations of Coccidioides spp. and the outbreaks of coccidiomycosis. Given the above, this study aimed at performing the molecular identification of 18 clinical and environmental isolates of C. posadasii, from Northeastern Brazil, maintained in the fungal collection of the Specialized Medical Mycology Center (CEMM), through PCR, as well as, to analyze the genetic diversity of these isolates by sequencing of 18S-28S regions of nuclear rDNA. The identification of the isolates was performed through PCR, using specific primers Coi9-1F and Coi9-R. The sequencing of the 18S-28S rDNA regions was performed through the method of chain termination by dideoxynucleotides, using the kit DYEnamicTM ET terminators cycle sequencing (GE Healthcare). The results confirmed the identification of all strains included in this study as belonging to the species C. posadasii. The phylogenetic tree based on 18S-28S rDNA region of C. posadasii from CEMM and Coccidioides spp. from Genbank. reveals the formation of a unique cluster encompassing the following strains CEMM 05-2-063, CEMM 05-2-064, CEMM 05-2-066 and CEMM 05-2-065, in a properly sustained branch, which apparently seems to group these isolates according to their geographical origin. The strains of C. posadasii showed lower genetic divergence in the ITS1 and ITS2 regions, when compared to strains of C. immitis. Analyses did not detect differences between strains of clinical origin and those of environmental origin. Further studies involving the analysis of fast evolving markers, such as microsatellites, can provide evidences to determine whether the groups found in this study are derived from a lineage of clonal reproduction. / A coccidioidomicose à uma infecÃÃo sistÃmica, predominantemente pulmonar, causada pelos fungos dimÃrficos e geofÃlicos, Coccidioides immitis e Coccidioides posadasii. No Brasil, a coccidioidomicose està associada a locais situados na zona semi-Ãrida da regiÃo Nordeste, considerada uma das Ãreas endÃmicas da doenÃa na AmÃrica do Sul. Estes patÃgenos consistem em espÃcies morfologicamente indistinguÃveis, mas que exibem diferenÃas moleculares peculiares. O DNA ribossÃmico nuclear (rDNA) de Coccidioides spp. tem sido apontado como importante marcador molecular utilizado na identificaÃÃo, taxonomia e filogenia. Atualmente, ainda hà escassez de mapas de abordagens epidemiolÃgicas para direcionar a correlaÃÃo entre as populaÃÃes de Coccidioides spp. com os surtos de coccidioidomicose. Diante do exposto, este estudo teve por objetivo, realizar a identificaÃÃo molecular de 18 isolados clÃnicos e ambientais de C. posadasii, oriundos do Nordeste brasileiro, mantidos na Micoteca do Centro Especializado em Micologia MÃdica (CEMM), atravÃs da tÃcnica de PCR, bem como, analisar a diversidade genÃtica destes isolados por meio do seqÃenciamento das regiÃes 18S-28S do rDNA nuclear. A identificaÃÃo dos isolados foi realizada por PCR utilizando os primers especÃficos Coi9-1F e Coi9-R. O sequenciamento das regiÃes 18S-28S rDNA foi realizado pelo mÃtodo da terminaÃÃo da cadeia pelo didesoxinucleotÃdeo, usando-se o kit DYEnamicTM ET terminators cycle sequencing (GE Healthcare). Os resultados confirmaram a identificaÃÃo de todas as cepas incluÃdas neste estudo, como pertencentes à espÃcie C. posadasii. A Ãrvore filogenÃtica, baseada na regiÃo 18S-28S rDNA de C. posadasii do CEMM, juntamente com sequÃncias de Coccidioides spp. depositadas no Genbank. revela a formaÃÃo de um cluster exclusivo englobando as cepas CEMM 05-2-063, CEMM 05-2-064, CEMM 05-2-065 e CEMM 05-2-066, em um ramo adequadamente sustentado, que aparentemente parece agrupar estes isolados segundo sua origem geogrÃfica. As cepas de C. posadasii apresentaram menor Ãndice de divergÃncia genÃtica nas regiÃes ITS1 e ITS2, quando comparadas Ãs cepas de C. immitis A anÃlise nÃo detectou diferenÃas entre as cepas de origem clÃnica e as de origem ambiental. Estudos posteriores envolvendo a anÃlise de marcadores de evoluÃÃo mais rÃpida, os microssatÃlites, podem fornecer evidÃncias para determinar se os agrupamentos encontrados neste estudo sÃo resultantes de uma linhagem de reproduÃÃo clonal.

FundaÃÃo de Amparo à Pesquisa do Estado do Cearà / A ordem Chiroptera mostra-se como reservatÃrio para vÃrios agentes infecciosos, incluindo protozoÃrios, bactÃrias, vÃrus e fungos. A fim de investigar a a eco-epidemiologia de Histoplasma capsulatum no Nordeste do Brasil, foram capturados 83 morcegos em Ãreas urbanas e rurais no estado do CearÃ, em coletas diurnas e noturnas, durante o perÃodo de agosto de 2010 a marÃo de 2011. ApÃs eutanÃsia, fragmentos de baÃo, fÃgado e pulmÃo foram removidos, macerados e alÃquotas de 100μL foram semeadas em placas contendo Ãgar BHI modificado, as quais foram incubadas por atà seis semanas nas temperaturas de 28ÂC e 35ÂC. AlÃquotas remanescentes de cada material macerado foram estocadas a -20ÂC. Embora o fungo nÃo tenha sido isolado em nenhuma amostra, apÃs 21 dias de incubaÃÃo à 35ÂC foi observado o crescimento de uma colÃnia fÃngica sugestiva de Coccidioides spp. em amostra de pulmÃo da espÃcie Carollia perspicillata. Fragmentos de pulmÃo do morcego retirados do estoque a -20ÂC e analisados ao microscÃpio Ãptico revelaram estruturas esfÃricas semelhantes a forma parasitÃria do fungo. A colÃnia foi investigada por meio do teste de reversÃo in vivo em modelo murino, com observaÃÃo dos animais inoculados por atà quatro semanas. A identificaÃÃo molecular das culturas foi realizada por reaÃÃo de PCR especÃfica para C. posadasii, empregando os oligonucleotÃdeos Coi9-R e Coi9-F. Por fim, amostras de macerados dos pulmÃes, baÃo e fÃgado foram investigadas quanto à presenÃa de anticorpos ou antÃgenos especÃficos para C. posadasii, bem como quanto à presenÃa de anticorpos anti-Histoplasma, por meio da tÃcnica de imunodifusÃo. EsfÃrulas caracterÃsticas de Coccidioides spp. foram visualizadas em amostras de pulmÃo dos camundongos infectados experimentalmente. AnÃlise molecular das culturas isoladas de pulmÃo de C. perspicillata, bem como dos ÃrgÃos dos camundongos, mostraram banda de aproximadamente 630 pb, caracterÃstica de C. posadasii. Por meio de reaÃÃo de imunodifusÃo, foram detectados dois animais reagentes para antÃgenos de Coccidioides. Os animais pertenciam Ãs espÃcies Glossophaga soricina e Desmodus rotundus e um animal da espÃcie G. soricina anticorpo-positivo para Coccidioides. NÃo foi observada presenÃa de anticorpos anti-Histoplasma em nenhuma das amostras. O presente estudo mostra o primeiro isolamento de C. posadasii em quirÃpteros. Adicionalmente, de forma inÃdita, detectou-se de antÃgenos e anticorpos especÃficos para C. posadasii nestes animais. Faz-se necessÃrio o estudo dos quirÃpteros na ecoepidemiologia do patÃgeno. / The order Chiroptera is shown as a reservoir for various infectious agents, including protozoan, bacteria, virus and fungi. To investigate the eco-epidemiological aspects of Histoplasma capsulatum in Northeast Brazil, 83 bats were captured in urban and rural areas in CearÃ, collected in day and night, during the period August 2010 to March 2011. After euthanasia, fragments of sleen, liver and lung were removed, macerated and 100 μL aliquots were plated on agar containing modified BHI, which were incubated for up to six weeks at temperatures of 28 ÂC and 35 ÂC. Aliquots of each remaing macerated material stored at -20 ÂC. Although the fungus was not isolated in any samples, after 21 days at 35 ÂC was observed the growth of a fungal colony suggestive of Coccidioides spp. in lung samples of the species Carollia perspicillata. Fragments of the bet lung removed from storage at -20 ÂC and analyzed by optical microscopy revealed spherical structures similar to the shape of the parasitic fungus. The colonies were investigated by means of the reversion in vivo in a murine model, with observation of the animals incoculated up to four weeks. The molecular identification of cultures was performed by PCR specific for C. posadasii, using the oligonucleotides Coi9-R and Coi9-F. Finally, samples of macerated lung, spleen and liver were investigated for the presence of antibodies or antigens specific for C. posadasii, as well as the presence of anti-Histoplasma, by immunodiffusion. Spherules of Coccidioides spp. features were visualized in lung samples of mice experimentally infected. Molecular analysis of cultures isolated from lungs of C. perpicillata, as well as organs of mice, showed approximately 630 pb band, characteristic of C. posadasii. By immunodiffusion reaction, two animals were detected reagents for Coccidioides antigens. The animals belonged to the species Glossophaga soricina and Desmodus rotundus and an animal of the species G. soricina antibody-positive for Coccidioides. There was no presence of anti-Histoplasma is any sample. The present study shows the first isolation of C. posadasii in bats. Additionally, in an unprecedented manner, we detected antigens and antibodies specific to C. posadasii these animals. Its is necessary to the study of bats in ecoepidemiology the pathogen.

Valley fever, or coccidioidomycosis, is a disease caused by the Coccidioides immitis and Coccidioides posadasii fungal species that dwell in the soil but can become airborne and infect a human or mammalian host through their respiratory tract. Disease rates in the western U.S. have significantly increased over the past two decades, creating an emerging public health burden. Studies have been conducted that attempt to elucidate the association between environmental conditions and the growth and dispersal of the pathogen, yet the specific ecology of and environmental precursors to the disease remain uncertain. This research project investigates the relationship between environmental variables and valley fever by modeling the spatial and temporal dynamics of the disease using varying techniques. Chapter 1 discusses relevant literature before discussing the challenges associated with studying valley fever. Chapter 2 analyzes the temporal relationships between valley fever and climatic variables, focusing on Kern County, California, an understudied region in the U.S. where valley fever is highly endemic. Chapter 3 focuses on a regional spatial analysis using ecological niche modeling to better understand the environmental factors that influence the overall spatial distribution of valley fever in the U.S. Finally, combining both spatial and temporal components, Chapter 4 uses a hierarchical Bayesian spatio-temporal model to investigate the patterns and drivers of this disease, focusing on state of California, which saw an approximate 200% increase in cases from 2014 to 2018. Cumulatively, this work offers new insights on relationships between climate, landcover, and valley fever disease risk. Significant findings include climate variables explaining up to 76% of valley fever variability in Kern County, California, the significance of both climatic and landcover variables in characterizing the geographic distribution of the disease, and identification of patterns increasing risk in geographic regions of California not currently considered highly endemic. These findings advance scholarly understandings of valley fever's environmental disease drivers. The results of this research can be applied by public health officials in the allocation of surveillance and public education resources, focusing upon regions that are most likely to encounter the illness. / Doctor of Philosophy / Valley fever is a fungal disease that causes illness in over ten thousand people in the western U.S. every year. Disease rates have been increasing for the past two decades for unknown reasons, although previous research suggests that climatic variations are likely contributing factors. This research evaluated environmental factors with hypothesized relationships to valley fever disease rates. First, this dissertation explored time-series relationships between climatic factors and valley fever incidence in an understudied county in California. Research findings identified that climatic factors including precipitation from previous seasons and temperature were significantly associated with valley fever incidence in this county. Second, this dissertation assessed where valley fever is found in the western U.S. The likely spatial distribution for the disease was mapped and environmental variables influential to this distribution were identified; they included both climate and landcover variables. Finally, a model was developed to analyze patterns of disease risk in California that considered both space and time, and environmental risk factors potentially contributing to the observed patterns were assessed. Counties with increased risk were identified and significant environmental relationships with valley fever risk were confirmed. The results of this research can be applied by public health officials in allocating surveillance and public education resources, focusing upon regions that are most likely to encounter the illness.

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / A coccidioidomicose e a histoplasmose sÃo micoses sistÃmicas que acometem o homem e animais, causadas por espÃcies de fungos dimÃrficos, com Ãnfase para Coccidioides posadasii e Histoplasma capsulatum var. capsulatum, respectivamente. SÃo consideradas doenÃas profundas importantes, podendo culminar em diversas complicaÃÃes secundÃrias. Nos Ãltimos anos, a melhoria dos mÃtodos de diagnÃstico micolÃgico e o aumento da ocorrÃncia de doenÃas imunossupressoras causaram grande impacto na incidÃncia das micoses profundas e oportunistas no mundo. Apesar da existÃncia de terapias eficazes com antifÃngicos contra a coccidioidomicose e a histoplasmose, a busca por novas drogas para o tratamento destas doenÃas se faz necessÃria. Ciprofloxacina à uma droga antibacteriana clÃssica do grupo das fluoroquinolonas, que inibe a atividade catalÃtica da DNA girase e topoisomerase IV, essenciais na replicaÃÃo e transcriÃÃo do DNA bacteriano. Estudos verificaram que ciprofloxacina pode atuar na DNA girase dos fungos. Assim, o presente estudo visou avaliar o efeito inibitÃrio in vitro de ciprofloxacina (CIP) isolada e em combinaÃÃo com os antifÃngicos anfotericina B (AMB), itraconazol (ITC), voriconazol (VRC) e caspofungina (CAS) frente à C. posadasii e Histoplasma capsulatum var. capsulatum. Foram utilizados 16 cepas de C. posadasii na fase filamentosa, 16 cepas de H. capsulatum var. capsulatum na fase filamentosa e 9 cepas de H. capsulatum var. capsulatum na fase leveduriforme. O estudo foi conduzido em ensaio de macrodiluiÃÃo e microdiluiÃÃo em caldo, descritos nos documentos M-38A e M-27A2, padronizados pelo Clinical Laboratory Standards Institute (CLSI), sendo utilizados para C. posadasii e Histoplasma capsulatum var. capsulatum, respectivamente. A interaÃÃo das drogas foi analisada atravÃs do cÃlculo do Ãndice da ConcentraÃÃo InibitÃria FracionÃria (FICI), definido como a soma das relaÃÃes entre a concentraÃÃo inibitÃria mÃnima (CIM) de cada droga em combinaÃÃo e a CIM da mesma droga isolada, considerando os valores menores ou iguais a 0,5 indicativos de sinergismo. Com relaÃÃo Ãs cepas de C. posadasii, foram observadas interaÃÃes sinÃrgicas em todas as combinaÃÃes, com destaque para as associaÃÃes de CIP (3,125≤CIM≤12,5 ug mL-1) com ITC (0,0078≤CIM≤0,125 ug mL-1) (n=13/16), CIP (3,125≤CIM≤12,5 ug mL-1) com VRC (0,0078≤CIM≤0,0312 ug mL-1) (n=13/16) e CIP (3,125≤CIM≤12,5 ug mL-1) com CAS (2≤CIM≤8 ug mL-1) (n=14/16). Para as cepas de H. capsulatum na fase filamentosa, tambÃm foram observadas interaÃÃes sinÃrgicas em todas as combinaÃÃes, com destaque para as associaÃÃes de CIP (3,906≤CIM≤62,5 ug mL-1) com ITC (0,00006≤CIM≤0,0078 ug mL-1) (n=14/16) e CIP (31,25≤CIM≤125 ug mL-1) com VRC (0,0156≤CIM≤0,125 ug mL-1) (n=16/16). No tocante Ãs cepas de H. capsulatum na fase leveduriforme, foram observadas poucas interaÃÃes sinÃrgicas nas combinaÃÃes de drogas testadas. Nenhuma das associaÃÃes de drogas testadas apresentou antagonismo. Os dados obtidos apontam uma nova alternativa para o tratamento da coccidioidomicose e da histoplasmose, sendo necessÃrios novos estudos que visem investigar os mecanismos de aÃÃo dessas combinaÃÃes de drogas no metabolismo celular fÃngico, bem como o delineamento de experimentos in vivo para confirmar a significÃncia desses achados. / Coccidioidomycosis and histoplasmosis are systemic mycoses that occur in humans and other animals and are caused by the dimorphic fungi Coccidioides posadasii and Histoplasma capsulatum var. capsulatum, respectively. They are considered important deep mycoses that can lead to several secondary complications. In the past years, the improvement of the techniques applied in mycological diagnosis and the increase in the occurrence of immunocompromising diseases have caused a great impact in the incidence of deep and opportunistic mycoses in the world. In spite of the existence of effective antifungal therapy against coccidioidomycosis and histoplasmosis, the pursue of new drugs to treat theses diseases is necessary. Ciprofloxacin is a classic antibacterial drug that belongs to the group of fluoroquinolones, which inhibit the catalytic activity of DNA gyrase and topoisomerase IV, which are essential in bacterial DNA replication and transcription. Some studies have shown that ciprofloxacin can act on fungal DNA gyrase. Thus, the present study aimed at evaluating the in vitro inhibitory effect of ciprofloxacin (CIP), when associated with amphotericin B (AMB), itraconazole (ITC), voriconazole (VRC) or caspofungina (CAS), on C. posadasii and H. capsulatum var. capsulatum. Sixteen strains of C. posadasii in the filamentous phase and 16 and 9 strains of H. capsulatum in the filamentous and yeast-like phase, respectively, were used. Broth macrodilution and microdilution assays were performed, as described in the documents M38-A and M27-A2, respectively, of the Clinical Laboratory Standards Institute (CLSI). Drug interaction was analyzed by calculating the fractional inhibitory concentration index (FICI), which is defined as the sum of the ratios between the minimal inhibitory concentration (MIC) of each combined drug and the MIC of the same drug isolatedly. Values of FICI smaller or equal to 0.5 indicate the occurrence of synergy. Concerning the isolates of C. posadasii, synergistic interactions were observed for all combinations, especially for the associations of CIP (3.125≤CIM≤12.5 ug mL-1) with ITC (0.0078≤CIM≤0.125 ug mL-1) (n=13/16), CIP (3.125≤CIM≤12.5 ug mL-1) with VRC (0.0078≤CIM≤0.0312 ug mL-1) (n=13/16) and CIP (3.125≤CIM≤12.5 ug mL-1) with CAS (2≤CIM≤8 ug mL-1) (n=14/16). For the isolates of H. capsulatum in the filamentous phase synergistic interactions were also observed for all combinations, with emphasis to the associations of CIP (3.906≤CIM≤62.5 ug mL-1) with ITC (0.00006≤CIM≤0.0078 ug mL-1) (n=14/16) and CIP (31.25≤CIM≤125 ug mL-1) with VRC (0.0156≤CIM≤0.125 ug mL-1) (n=16/16). For H. capsulatum in yeast-like phase, few synergistic interactions were observed for the tested drug combinations. None of the tested combinations presented antagonism. The obtained data may point at a new alternative for the treatment of coccidioidomycosis and histoplasmosis. Thus, it is necessary to investigate the mechanisms of action of these drug combinations on the fungal cellular metabolism and to perform in vivo experiments to confirm the relevance of these findings.

Thesis (Ph.D.)--Medical University of Ohio, 2006. / "In partial fulfillment of the requirements for the degree of Doctor of Philosophy in Medical Sciences." Major advisor: Garry Cole. Includes abstract. Document formatted into pages: ii, 206 p. Title from title page of PDF document. Title at ETD Web site: Evaluation of two homologous Coccidioides posadasii antigens as recombinantly expressed monovalent, divalent, and chimeric vaccine candidates. Bibliography: pages 75-83, 116-120, 165-169, 185-204.

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / PeptÃdeos bioativos naturais sÃo substÃncias encontradas em diversas espÃcies que podem alterar a resposta imune contra patÃgenos, seja de forma prejudicial ou proteÃÃo para o hospedeiro. Hà um crescente interesse na utilizaÃÃo de produtos naturais para a modulaÃÃo de processos inflamatÃrios, alguns deles derivados de fungos nÃo patogÃnicos. Fungos patogÃnicos, no entanto, tambÃm apresentam componentes imunomoduladores. Estudos prÃvios mostraram que extratos obtidos de nematÃdeos modularam a resposta imune em modelo experimental. Seguindo a mesma linha de pesquisa, foi investigado o efeito de um extrato obtido do fungo Coccidioides posadasii em modelo de artrite induzida por zymosan (AZy). Ratos e camundongos receberam 1 mg e 0,1 mg de zymosan intra-articular (i.a.), respectivamente. Grupos receberam o extrato de C. posadasii por via oral (v.o.) ou intraperitoneal (i.p.) 30 min antes do zymosan i.a. Grupos-controle receberam apenas soluÃÃo salina. A hipernocicepÃÃo foi medida utilizando o teste de incapacitaÃÃo articular e o von Frey eletrÃnico. O influxo celular agudo e crÃnico, bem como os nÃveis de nitrito e citocinas, foram avaliados no exudato sinovial. A sinÃvia foi utilizada para exame histopatolÃgico. O dano na cartilagem foi avaliado mediante determinaÃÃo de conteÃdo de glicosaminoglicanos (GAG). O prÃ-tratamento com o extrato de C. posadasii, seja i.p. ou v.o., inibiu significativamente e de forma dose-dependente, o influxo de cÃlulas tanto na fase aguda como na fase crÃnica, bem como a hipernocicepÃÃo, acompanhada por uma ligeira reduÃÃo na perda de GAG e uma melhora significativa da sinovite crÃnica. ReduÃÃo e alquilaÃÃo do extrato reverteram os efeitos protetores observados anteriormente. A administraÃÃo do extrato de C. posadasii nÃo alterou os nÃveis i.a. de NO, IL-1β, TNF-α, IFN-γ e IL-17 em ratos e camundongos submetidos à AZy, enquanto nÃveis de IL-10 foram significativamente reduzidos. Os dados revelam que um extrato de C. posadasii reduz a expressÃo de iNOS, que està associado com a inibiÃÃo da apoptose sinovial e diminuiÃÃo dos nÃveis de IL-10 liberados. Esses dados mostram um papel anti-inflamatÃrio sistÃmico e imunomodulador para o extrato de C. posadasii em AZy. Uma abordagem preliminar, utilizando tÃcnicas eletroforÃticas, para caracterizar componentes ativos excluiu carboidratos carregados enquanto aponta para fraÃÃes proteicas ou polipeptÃdeo como responsÃveis pela atividade biolÃgica. O efeito do extrato de C. posadasii à espÃcie-independente. CaracterizaÃÃo adicional dos componentes ativos no extrato pode revelar mecanismos relevantes para compreender a resposta do hospedeiro contra os componentes fÃngicos. AlÃm disso, o efeito anti-inflamatÃrio do extrato ilustra a importÃncia da realizaÃÃo de estudos, uma vez que a atividade oral pode tambÃm ser relevante para o tratamento de doenÃas inflamatÃrias. / Bioactive Natural Peptides are substances found in diverse species and may alter the immune response against pathogens, being either protective or harmful to the host. There is a growing interest in using natural products for the modulation of inflammatory processes, some of them derived from non-pathogenic fungi. However, pathogenic fungi also have immunomodulatory components. Previous studies showed that extracts of nematodes modulate the immune response in experimental model. Following the same line of research, it was investigated the effect of a protein-rich extract from Coccidioides posadasii in model of zymosan-induced arthritis (ZYA). Rats and mice received 1 mg and 0.1 mg zymosan intra-articularly (i.a.), respectively. Test groups received C. posadasii extract either per os or intraperitoneally (i.p.) 30 min prior to zymosan i.a. Controls received saline. Hypernociception was measured using the articular incapacitation test and the von Frey electronic test. Cell influx, nitrite, and cytokines levels were assessed in joint exudates. The synovia was used for histopathology. Cartilage damage was assessed through determining glycosaminoglycan (GAG) content. Pretreatment with the C. posadasii extract, either i.p. or per os, significantly and dose-dependently inhibited both acute and chronic cell influx as well as hypernociception, with a mild reduction of GAG loss and significant amelioration of the chronic synovitis. Reduction and alkylation of the extract abrogated protector effects seen previously. Administration of the C. posadasii extract did not alter i.a. levels of NO, IL-1β, and TNF-α in rats subjected to ZYA, whereas intra-articularly levels of IL-10 were significantly reduced. Data reveal that a C. posadasii extract reduces iNOS expression that is associated to inhibition of synovial apoptosis and decrease of IL-10 levels released into zymosan-inflamed joints.These data show a systemic anti-inflammatory and immunomodulatory role for the C. posadasii extract in ZYA. A preliminary approach, using electrophoresis, to characterize active components excluded the presence of carbohydrates while pointing to a protein or polypeptide present in extract as responsible for the biological activity. The protective effect of the C. posadasii extract is species-independent. Further characterization of the active components in this extract may unravel mechanistic effects relevant to understand the host response against fungal components. In addition, the anti-inflammatory effect of the extract illustrates the relevance of pursuing studies, since the oral activity may also be of relevance to the treatment of inflammatory diseases.