Rozprawy doktorskie na temat „Toxoid”

This thesis describes the immunization protocols for the production of antibodies against tetanus toxin and toxoid in guinea pigs and mice. Antibodies were successfully raised against the toxin without mortalities in either species. The murine sera obtained, were isotyped by ELISA and the toxin was proven to be a superior antigen in eliciting production of IgG$\rm\sb{2a}$ and IgG$\sb3$. The two isotypes which have demonstrated antitumor activity. The anti-toxoid sera exhibited a lower reactivity towards the toxin and toxoid when compared with anti-toxin sera. The reactivity of recombinant tetanus toxin fragment C was studied and the results indicated that in the murine serum, 72% of anti-toxin or anti-toxoid antibodies were directed against epitopes on fragment C. The study of the guinea pig sera suggested that similar to mouse serum, it can develop in response to toxin as an antigen, antibodies against toxin which are mostly directed against the fragment C portion. On the other hand. guinea pigs seem to respond to the toxoid as an antigen by producing antibodies to more than fragment C. (Abstract shortened by UMI.)

This study examines the possible mechanisms by which radiation and the bacterial toxin, Tetanus toxoid (TT), suppress the murine splenic mononuclear cell (SMNC) response to mitogen. This study demonstrates that the lymphocyte proliferation response of SMNC to the mitogenic lectin PHA can be suppressed by TT in a dose-dependant manner in vitro, without affecting the viability of the cells, in the anti-proliferative concentrations used (0.5-5 $\mu$g/ml). SMNC pre-incubated with TT could suppress the pHA blastogenic response of fresh autologous cells during co-incubation suggestion the involvement of activated suppressor cells. Flow cytometric analysis demonstrated that TT does not produce an alteration in the cellular balance, indicating that the suppression would appear to be dependant upon a change in T cell function. TT down-regulated the expression of class II MHC antigens on antigen-presenting cells which may represent an inappropriate costimulatory signal required for T cell activation. Whole body irradiation has been reported to induce active immune suppression. In the present study, ionizing radiation (0-700 cGy) produced decreased spleen cellularity and decreased ability of surviving SMNC to respond to mitogen. There was no evidence, however, to indicate that irradiation (100 cGy) activated suppressor cells during the first 7 days post-irradiation. Similarly, radiation did not seem to interact with TT to increase the amount of TT-induced suppression. (Abstract shortened by UMI.)

Previous studies have shown that certain antigens can down-regulate immune responses of human peripheral blood mononuclear cells (PBMC) both in vivo and in vitro. This study demonstrates that tetanus toxoid (TT), in a dose-dependent fashion, suppresses the induction of a blastogenic response of PBMC by phytohemagglutinin (PHA), monoclonal anti-CD3, and anti-CD4 antibody. Pokeweed mitogen (PWM)-induction of IgG and IgM is suppressed as well. The suppression is partially reversed by indomethacin and IL-2, but not by IL-1 or tumor necrosis factor (TNF-$\alpha$). PBMC pre-incubated with TT could suppress the PHA blastogenic response of fresh autologous cells during co-incubation. The removal of CD4$\sp+$ cells prior to induction of suppression greatly diminished the suppression of PHA blastogenic response, whereas the elimination of CD8$\sp+$ cells had no effect. Therefore it is concluded that TT induces non-specific suppressor cells that can strongly dampen mitogenic responses, and that CD4$\sp+$ cells plays an important role in this suppression. CD4 $\sp+$ cell, together with monocytes, may suppress mitogenic responses involving a prostaglandin-dependent pathway.

The ƒÑ-toxin of Clostridium perfringens is a toxin involved in numerous diseases of humans and agriculturally important animals. One of these diseases is necrotic enteritis (NE), a sporadic enteric disease which affects avian species world-wide. This study involved the inactivation of alpha-toxin (ƒÑ-toxin) for use as a potential vaccine candidate to combat NE in chickens, and other diseases caused by C. perfringens type A. During the course of this research a number of ƒÑ-toxin recombinant proteins were developed through molecular inactivation of the ƒÑ-toxin gene, plc. Proteins plc316 and plc204 were developed by the deletion of the first three and seven ƒÑ-helices of the N-terminal domain respectively. These deletions resulted in proteins which were unstable in solution, constantly aggregated into insoluble masses and elicited lower overall antibody responses when administered to mice. A third protein, plcInv3 was developed from the deletion of part of the catalytic domain of the ƒÑ-toxin. PlcInv3 was highly soluble and upon immunisation of mice elicited a significant antibody response which was also capable of protecting mice against a live challenge of C. perfringens. The fourth and final protein developed was plc104. The smallest of the recombinant ƒÑ-toxin proteins, it consisted entirely of the C-terminal domain of ƒÑ-toxin. Its small size did not affect its ability to induce a strong antibody response when administered to mice, the antibodies of which were also protective during a challenge with C. perfringens. STM1, an attenuated strain of S. Typhimurium was used in the development of a vectored vaccine for the expression and oral delivery of plcInv3 and plc104 within the mouse host. The proteins were expressed within STM1 from expression plasmids containing the in vivo inducible promoters PhtrA and PpagC. A measurable humoral immune response against ƒÑ-toxin was absent following three oral vaccinations with the vectored vaccines, although, cytokine profiling of splenocytes from vaccinated mice revealed an increase in the number of interleukin-4 (IL-4)secreting cells and the lack of interferon-gamma (IFN-ƒ×) secreting cells. This indicated the stimulation of a T-helper type 2 (TH2) immune response which also lead to partial protection against a live C. perfringens challenge. This study demonstrates the feasibility of using STM1 as a carrier for the in vivo expression of the C. perfringens ƒÑ-toxin recombinant proteins plcInv3 and plc104. It is the first study to express C. perfringens antigens within an attenuated strain of S. Typhimurium, STM1.The partial protection of mice immunised with these vaccines indicates there is potential for this vectored vaccine system to be used in the protection of diseases caused by the ƒÑ-toxin of C. perfringens.

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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)
Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)
Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
A quitosana (Chi), a (1-4)-amino-2-desoxi-ƒÒ-glicana, e a forma desacetilada da quitina, um polissacarideo das conchas de crustaceos. As suas caracteristicas unicas como a carga positiva, biodegradabilidade, biocompatibilidade, atoxicidade e estrutura rigida fazem com que esta macromolecula seja ideal para uso como sistema oral de entrega de vacinas. Foram preparadas vesiculas unilamelares grandes (REVs) envoltas por dentro e por fora (como um sanduiche) com quitosana (Chi) e poli-vinil alcool (PVA). Entretanto, existem alguns problemas as serem superados com relacao a estabilizacao da proteina durante este processo. Durante a fase de formacao de micelas reversas, no processo de nanoencapsulacao da proteina, expandem-se as interfaces hidrofobicas que entao levam as adsorcoes interfaciais seguidas por desenovelamento e agregacao das proteinas. Aqui, observaram-se atraves de tecnicas espectroscopicas e imunologicas, o uso dos sais da serie de Hoffmeister durante a fase de formacao de micela reversa para estudar a conformacao estavel do toxoide difterico (Dtxd). Foi estabelecida uma correlacao entre os sais usados na fase aquosa e as variacoes na solubilidade e conformacao de Dtxd. Como o conteudo em helice-ƒÑ foi praticamente estavel concluiu-se que a encapsulacao de Dtxd ocorreu sem agregacao ou sem exposicao de residuo hidrofobico na proteina. A agregacao de Dtxd foi evitada em 98 % quando se usou o cosmotropico PO2-4. Este ion foi usado para se preparar uma formulacao de Dtxd em REVs-Chi-PVA estavel e com identidade imunologica reconhecida na presenca de PO2-4. Entao, obteve-se uma solubilidade e estabilidade maxima de Dtxd depois de seu contacto com CH3CO2C2H5 para comecar a sua nanoencapsulacao em condicoes ideais. Este foi um avanco tecnologico importante porque uma solucao simples, como e a adicao de sais, evitou o uso de proteinas heterologas (Rescia et alii, 2009a). A proteina estabilizada foi entao encapsulada dentro de REVs como o descrito. Os lipossomas tem sido descritos como adjuvantes desde 1974 (Allison e Gregoriadis, 1974). A maior limitacao de seu uso em vacinas orais e a sua instabilidade estrutural causada pelas atividades enzimaticas do meio. O objetivo aqui foi combinar lipossomas, que podem encapsular antigenos (Dtxd, Diphtheria toxoid) com quitosana que protege estas particulas e promove a mucoadesibilidade. Empregaram-se tecnicas fisicas para se entender o processo pelo qual lipossomas (SPC: Cho, 3: 1) podem ser recobertos (interna e externamente) com quitosana (Chi) e PVA (poly-vinilic-alcohol) que sao polimeros biodegradaveis e biocompativeis. Obtiveram-se particulas de REVs-Chi (vesiculas preparadas por evaporacao de fase reversa recobertas interna e externamente com Chi) redondas e com as superficies rugosas e estabilizadas ou nao com PVA. As eficiencias de encapsulacao (Dtxd foi usada como antigeno) foram diretamente dependentes da presenca de Chi e PVA na formulacao. A adsorcao de Chi a superficie de REVs foi acompanhada por um aumento no potencial ƒê. Em contraste, a adsorcao de PVA a surperficie de REVs-Chi foi acompanhada por uma diminuicao do potencial . A presenca de Dtxd aumentou a eficiencia de adsorcao de Chi as superficies. A afinidade de PVA pela mucina foi 2000 vezes maior do que a observada somente com Chi e nao depende se a molecula esta em solucao ou se esta adsorvida a superficie lipossomal. A liberação do Dtxd foi retardada por sua encapsulação dentro de REVs-Chi-PVA. Concluiu-se que estas novas vesículas estabilizadas foram hábeis em se adsorverem às superfícies intestinais, resistiram às degradações e controlaram a liberação do antígeno. Assim, as partículas de REVs-Chi-PVA podem ser usadas como um veículo oral com capacidade adjuvante (Rescia et alii, 2009b). Os lipossomas revstidos por quitosana (REVs-Chi) como veículos orais para transporte de vacinas foram bem caraterizados neste laboratório. Estas partículas foram desenhadas para serem capturadas pelo muco, para interagirem com surperfícies orais e para resistirem às enzimas do trânsito gástrico. Foram usadas três formulações diferentes contendo o Dtxd (toxoide diftérico) para imunizar camundongos: REVs [Vesículas unilamelares obtidas por evaporação de fase reversa produzidas com SPC: Cho (3:1)]; REVs-Chi (REVs recobertas por Chi) e REVs-Chi-PVA (REVs recobertas por Chi e estabilizadas por PVA). Através do teste de adesibilidade e dos experimentos com anti-toxoide diftérico observou-se que houve uma correlação direta entre a complexidade da partícula (antígeno livre < REVs < REVs-Chi < REVs-Chi-PVA) e a produção de anticorpos (IgA, IgG1 and IgG2a) em todos os ensaios (R= 0,91766- 0,99718). O resultado mais interessante foi a total ausência da produção de IgA nos camundongos imunizados com o antígeno livre, provando então a excelência das partículas engenheiradas. Além do aumento da produção dos anticorpos de mucosa, ambas formulações com Chi ou com Chi-PVA estimularam tanto a produção de anticorpos humorais quanto a seletividade. Demonstrou-se que é possível de se estabelecer uma correlação entre REVs-Chi/Dtxd and REVs-Chi-PVA/Dtxd e o aumento da imunidade de mucosa. Estas partículas podem ser usadas como veículo geral tanto para transporte de drogas quanto de vacinas (Rescia et alli, 2009c).
Chitosan, - (1-4)-amino-2-deoxy-D-glucan) is a deacetylated form of chitin, a polysaccharide from crustacean shells. Its unique characteristics such as positive charge, biodegradability, biocompatibility, non-toxicity, and rigid structure make this macromolecule ideal for oral vaccine delivery system. We prepared reverse phase evaporation vesicles (REVs) sandwiched by chitosan (Chi) and polyvinylic alcohol (PVA). However, in this method there are still some problems to be circumvented related to protein stabilization. During the inverted micelle phase of protein nanoencapsulation, hydrophobic interfaces are expanded leading to interfacial adsorption followed by protein unfolding and aggregation. Here, spectroscopic and immunological techniques were used to ascertain the effects of the Hoffmeister series ions on Diphtheria toxoid (Dtxd) stability during the inverted micelle phase. A correlation was established between the salts used in aqueous solutions and the changes in Dtxd solubility and conformation. Dtxd α-helical content was quite stable what led us to conclude that encapsulation occurred without protein aggregation or without exposition of hydrophobic residues. Dtxd aggregation was 98 % avoided by the kosmotropic PO2-4. This ion was used to prepare a stable Dtxd and immunologically recognized REVs-Chi-PVA formulation in the presence of 50 mM PO42-. Under these conditions the Dtxd retained its immunological identity. Therefore, we could obtain the maximum Dtxd solubility and stability after contact with CH3CO2C2H5 to begin its nanoencapsulation within ideal conditions. This was a technological breakthrough because a simple solution like salt addition avoided heterologous proteins usage (Rescia et al., 2009a). The stabilized protein was as encapsulated within REVs as described. Liposomes have been used as adjuvants since 1974 (Allison and Gregoriadis, 1974). One major limitation for the use of liposomes in oral vaccines is the lipid structure instability caused by enzyme activities. Our goal was to combine liposomes which can encapsulate antigens (Dtxd, diphtheria toxoid) with chitosan which protects the particles and promotes mucoadhesibility. We employed physical techniques to understand the process by which liposomes (SPC: Cho, 3:1) can be sandwiched with chitosan (Chi) and stabilized by PVA (Poly-vinylic alcohol) which are biodegradable and biocompatible polymers. Round and smooth surfaced particles of REVs-Chi (Reversed phase vesicles sandwiched by Chi) stabilized by PVA were obtained. The REVs encapsulation efficiencies (Dtxd was used as the antigen) were directly dependent on the Chi and PVA present in the formulation. Chi adsorption on REVs surface was accompanied by an increase of  otential. In contrast, PVA adsorption on REVs-Chi surface was accompanied by a decrease of potential. The presence of Dtxd increased the Chi surface adsorption efficiency. The PVA affinity by mucine was 2000 higher than that observed with Chi alone and did not depend on the molecule being in solution or adsorbed on the liposomal surface. The liberation of encapsulated Dtxd was retarded by encapsulation within REVs-Chi-PVA. These results lead us to conclude that these new and stabilized particles were to able to adsorb to intestinal surfaces, resisted degradation and controlled the antigen release. Therefore, REVs-Chi-PVA particles can be used as an oral delivery adjuvant (Rescia et al., 2009b). Liposomes sandwiched by chitosan (REVs-Chi) as vehicles for oral vaccines have been well characterized in our laboratory. These particles were designed to be captured by mucus, to interact with oral surfaces and to withstand the enzymes of the gastric transit. Three different formulations containing Dtxd (diphtheria toxoid): REVs [reverse phase evaporation vesicles of SPC: Cho (3: 1)]; REVs-Chi (REVs sandwiched by chitosan) and REVs-Chi-PVA were used to immunize mice. Through adhesibility assays and antibody anti-diphtheria experiments we observed a direct correlation between particle complexity (free antigen < REVs < REVs-Chi < REVs-Chi-PVA) and antibody production (IgA, IgG1 and IgG2a) in all the assays (R= 0,91766- 0,99718). The most striking result was the absence of IgA production in those mice immunized with the free antigen, proving the excellence of the engineered particles. In addition to enhancement of mucosal antibodies production, the formulations with Chi and PVA stimulated both, humoral antibody production and selectivity. We have shown that it was possible to establish a correlation between REVs-Chi/Dtxd and REVs-Chi-PVA/Dtxd and the enhancement of mucosal immunity. These particles can be used as a general vehicle for oral drug or vaccine delivery systems (Rescia et al., 2009c).
TEDE
BV UNIFESP: Teses e dissertações

The incidence and mortality rates of neonatal tetanus (NNT) remain underreported in Nigeria. This cross-sectional study was guided by the Mosley and Chen's model for the elements of child survival in developing countries. The goals of the study were twofold: (a) to compare the NNT prevalence and the mortality rates from the existing surveillance system and active surveillance of health facility records in 7 selected health facilities from 2010 to 2014 in Katsina state, Nigeria and (b) to assess the associations between selected NNT risk factors, number of maternal tetanus toxoid injections, frequency of antenatal visits, place of delivery, and cord care, and neonatal mortality as the outcome variable. Data from 332 NNT records were extracted through retrospective records review and analyzed using a logistic regression model. The prevalence of NNT and mortality rate were 336 cases and 3.4 deaths per 100,000 population, respectively, while the prevalence of NNT and mortality rate reported through the IDSR system were 111 cases and 1.0 death per 100,000 population, respectively. Only neonates whose mothers had 1 dose of tetanus toxoid vaccine were significantly associated with NNT mortality, (p < 0.05), OR = 4.12, 95% CI [1.04, 16.29]. Frequency of antenatal visits, place of delivery, and cord care were all not significant predictors of NNT mortality. Implications for positive social change include gaining knowledge on associations between NNT risk factors and neonatal mortality, and strengthening the NNT surveillance system with the capacity for early detection of potential risk factors to develop specific public health interventions aimed at improving the outcome of neonatal tetanus.

N.meningitidis é diplococcus gram-negativo, patógeno estritamente humano que similarmente a outras bactérias é circundado por membrana externa, com lipídios, proteínas (OMP) e lipopolissacárides. Ela tem sido uma das principais causas da meningite e de outras infecções invasoras no mundo. Este trabalho buscou usar o toxóide STX2 de E.coli como adjuvante para um possível e futuro modelo vacinal e como estimulante antigênico, proteínas da membrana externa do meningococo (OMP) transportados em lipossomas. Observaram-se diferenças na produção de anticorpos IgG obtidas entre os camundongos após cada uma das 3 sangrias mas, não quanto ao índice de avidez. A nova preparação antigênica desencadeou um alto título, mesmo após um ano da 1ª imunização, estimulou a produção de anticorpos para outros sítios de ligação e serviu como proteção ao LPS residual dos processos com deoxicolato da OMP, diminuindo toxicidade da preparação IM reduzindo os riscos para idosos e crianças muito pequenas e também, em imunizações de longo termo, com grande vantagem aos sistemas tradicionais.
N.meningitidis is diplococcus gram-negative strict human patogen that similarly to other bacteria are surrounded by external membrane with lipids, proteins (OMP) and LPS. It has been one of the main causes of the meningitidis and other invading infections in the world. This work searched to use STX2 toxoid of E.coli as adjuvant for a possible and future vaccine model and as antigenic stimulant proteins of the external membrane of meningococci (OMP) carried in liposomes. Differences in the production of IgG antibodies gotten between the mice each one of the 3 bleedings had been observed after but not how much to the avidity index. The new antigenic preparation unchained one high heading exactly after one year of 1st immunization stimulated the production of antibodies for other sites of linking and served as protection to the residual LPS of the processes with deoxicolate of the OMP diminishing toxicity of IM preparation reducing the aged risks for and very small children e also, in immunizations of long term with great advantage to the traditional systems.

Vaccination plays a very important part in daily life. It is essential to get vaccinated at an early age. The conventional parented method used is not always effective and not cost efficient. It requires qualified personnel and sterile conditions for administration of the vaccines. The aim of this study was to investigate the effect of chitosan, N-trimethyl chitosan chloride (TMC) and Emzaloid™ particles on the local and systemic immune response of mice after oral vaccination with Diphtheria toxoid (DT). The different formulations used were chitosan microparticles (± 10 µm), chitosan nanoparticles (± 400 nm), TMC microparticles (± 5 µm), Emzaloid microparticles (± 4 µm) and Emzaloid nanoparticles (± 500 nm). All of these formulations proved to be very good delivery systems and can entrap large amounts of the antigen. Balb/c mice were used to determine the local and systemic immune response of these formulations. The mice were vaccinated orally on three consecutive days in week 1 and 3 with 40 Lf DT per week with a total volume of 300 µl. Blood samples were taken from the mice and analysed for a systemic immune response (IgG). The same mice were used to determine the local immune response (IgA). Faeces were collected from each mouse on day 1, 3, 4, 6, 14 and 20 for analysis. An enzyme-linked immunosorbent assay (ELISA) was used to determine IgG and IgA titers. It can be concluded that chitosan nanoparticles was the only formulation with a higher response than that of the currently used vaccine. Emzaloid nanoparticles showed no significant difference in response when compared to the currently used vaccine. All the other formulations showed a much smaller response than that of the conventional method of vaccination.
Thesis (M.Sc. (Pharmaceutics))--North-West University, Potchefstroom Campus, 2005.

Since 2006, Uganda has experienced a nonchanging neonatal mortality rate of 27 out of 1,000 live births, which is higher than the global average of 19 deaths for every 1,000 live births. The purpose of this retrospective cross-sectional study was to determine factors affecting access to health services and their impact on newborn deaths in Uganda. Mosley and Chen's model for child survival in developing nations provided the framework for the study. Secondary data from the 2016 demographic and health survey (UDHS) collected by the Uganda Bureau of Statistics (UBOs) was used. A total of 7,538 cases were used and analyzed using binary logistic regression and one-way analysis of covariance (ANCOVA). The results showed attending less than 4 antenatal care (ANC) visits during pregnancy increased the odds of neonatal deaths 1.57 times, while not taking antimalarial drugs during pregnancy increased the odds of neonatal deaths 1.67 times. However, receiving 4 or more tetanus toxoid (TT) vaccine doses before pregnancy was not statistically associated with an increased risk of neonatal death (p = .597). Also, there was no significant relationship between neonatal mortality and whether distance to health facilities was a challenge (p = .276) or receiving medical assistance during childbirth (p = .420). While there were significant differences in deaths of newborns in geographic regions while controlling for the number of ANC visits (p = .023), there were no differences while controlling for all three covariates, F(4, 117) = 2.00, p = .098. Findings may be used to inform government policies on ANC and malaria prevention during pregnancy, which may reduce neonatal mortality rates in Uganda.

Previous studies have demonstrated that chitosan and its derivative, N-trimethyl chitosan chloride (TMC) are effective and safe absorption enhancers to improve mucosal delivery of macromolecular drugs including vaccines. Furthermore, chitosan and TMC can easily form microparticles and nanoparticles, which have the ability to encapsulate large amounts of antigens. Emzaloid™ technology has proven in the past to be an effective delivery system for numerous drugs. Emzaloids can entrap, transport and deliver large amounts of drugs including vaccines. In this study, the ability of chitosan microparticles and nanoparticles, TMC microparticles as well as micrometer and nanometer range Emzaloids to enhance both the systemic and mucosal (local) immune response against diphtheria toxoid (DT) after nasal administration in mice was investigated. The above mentioned formulations were prepared and characterised according to size and morphology. DT was then associated to the chitosan microparticles and nanoparticles as well as TMC microparticles to determine the antigen loading and release. It was found that the loading efficacy of the formulations was 88.9 %, 27.74 % and 63.1 % respectively, and the loading capacity of the formulations was 25.7 %, 8.03 % and 18.3 %. DT loaded and unloaded (empty) chitosan microparticles and nanoparticles, TMC microparticles, micrometer and nanometer range Emzaloids as well as DT in phosphate buffered saline (PBS) were administered nasally to mice. Mice were also vaccinated subcutaneous with DT associated to alum as a positive control. All mice were vaccinated on three consecutive days in week 1 and boosted in week 3. Sera was analysed for anti- DT IgG and nasal lavages were analysed for anti-DT IgA using an enzyme linked imrnunosorbent assay (ELISA). In the study conducted to determine the systemic (IgG) and local (IgA) immune responses it was seen that DT associated to all the experimental formulations produced a systemic immune response. The said formulations produced a significantly higher systemic immune response when compared to the formulation of DT in PBS. Furthermore, the mice vaccinated with DT associated to the TMC formulations showed a much higher systemic immune response than the mice that were vaccinated subcutaneously with DT associated to alum, whereas the other formulations produced systemic immune responses that were comparable to that of DT associated to alum. It was also found that DT associated to the experimental formulations produced a local immune response, however only DT associated to TMC microparticles produced a consistent local immune response. It can be concluded from the in vivo experiments that the TMC formulations, moreover, the TMC microparticles is the most effective and promising formulation for the nasal delivery of vaccines.
Thesis (M.Sc. (Pharmaceutics))--North-West University, Potchefstroom Campus, 2005.

Toxic shock syndrome toxin-1 (TSST-1), an exoprotein produced by some strains of Staphylococcus aureus, is implicated in the pathogenesis of menstrual TSS. However, its role in nonmenstrual TSS is less certain. In order to study the pathogenetic role of TSST-1 in TSS, three approaches were taken: a) to develop an ELISA for detection of TSST-1 in biologic fluids in order to verify TSST-1 production in vivo in TSS patients, b) to quantitate TSST-1 specific antibodies in the serum of TSS patients and controls to determine whether such antibodies are protective, and c) to attempt to identify other staphylococcal products which may be implicated in some forms of TSS. A sensitive and specific noncompetitive enzyme-linked immunosorbent assay (ELISA) capable of detecting TSST-1 at concentrations from 0.5 to 16 ng/ml was developed. This assay did not detect other staphylococcal enterotoxins including A, B, C₁, C₂, C₃, D and E. Possible interference by protein A was readily eliminated by pretreatment of test samples with 10% nonimmune rabbit serum. The assay was adapted for rapid screening of TSST-1 production by S. aureus isolates in culture supernatants in vitro, and for the detection of TSST-1 in vaginal washings and urine of TSS patients and healthy controls in vivo. All 35 S. aureus isolates confirmed to be TSST-1 positive by Ouchterlony immunodiffusion, and 59 of 60 isolates confirmed to be TSST-1 negative, gave concordant results by ELISA. Interestingly, toxigenic S. aureus strains isolated from TSS patients quantitatively produced significantly more toxin in vitro compared to toxigenic control strains (p<0.05, Mann-Whitney rank sum test). TSST-1 could be detected by ELISA in 3 of 4 vaginal washings collected within 3 days of hospitalization from 3 women with acute menstrual TSS, compared to 0 of 17 washings from 9 TSS women collected greater than 3 days after hospitalization (p=0.003, Fisher's exact test) and 1 of 15 washings from 14 healthy control women (p=0.016). TSST-1 was not detected in the urine of 4 acute TSS patients, 2 convalescent TSS patients or in 3 control urine tested. A sensitive and reproducible ELISA was also developed for the quantitation of TSST-1 specific IgG in serum. Anti-TSST-1 was assessed in acute and convalescent sera from 16 nonmenstrual (9 female, 7 male) and 14 menstrual TSS patients, and from 87 healthy women and 66 healthy men as controls. Quantitative levels of anti-TSST-1 in the study groups were calculated as the percent of standard activity (POSA) relative to a medium titre reference serum standard. ELISA titers in acute sera from menstrual TSS (26.2 ± 5.2, mean POSA ± S.E.M.), but not nonmenstrual TSS women (71.8 ± 18.6), were significantly lower than in healthy controls (78.9 ± 7.3) (p<0.01, Mam-Whitney test). Titers from menstrual TSS patients remained low (25.2 ± 10.7) even during late convalescence (mean duration 20 months after illness onset), compared to healthy female controls (p<0.05). Acute titers in males with TSS (37.0 ± 15.6) were also significantly lower than those in control men (114.6 + 11.0) (p<0.05). An inverse relationship of recovery of toxigenic S. aureus and anti-TSST-1 titers in acute sera of TSS patients was observed. Interestingly, antibody titers in control men were significantly higher than in control women (p<0.001). No age-dependent effects or interactive effects of age and sex on ELISA titers were observed. To enable immunoblot analyses, TSST-1 was produced and partially purified using column chromatography techniques. Percent recovery of TSST-1 from culture supernatant through to the final procedure was approximately 15.5%. The relative purity of TSST-1 (TSST-l/total protein, w/w) was increased from 0.21% in culture supernatants to 94.4% in the final product. Ouchterlony immunoprecipitation against reference rabbit antitoxin demonstrated identity with reference TSST-1 as well as with TSST-1 prepared in other laboratories. Physical characterization demonstrated a molecular weight of 24 kd and a pi of 7.0. Using pooled normal human serum as a first antibody probe, several bands in addition to the 24 kd TSST-1 band were visualized by immunoblot against our partially purified toxin as well as similar preparations obtained from other investigators. To determine whether any of the additional bands might be implicated in TSS, acute and convalescent sera from TSS patients were used to probe for immunoreactive bands in our partially purified TSST-1 as well as a commercially obtained preparation. Seroconversion was demonstrated to the 24 kd TSST-1 protein in 7 of 10 TSS patients from whom toxigenic S. aureus was isolated. In addition, seroconversion was noted to a 49 kd band in 4 patients, to a 21 kd band in 3 patients, to a 28 kd band in 1 patient and to a 32 kd band in 2 patients. In conclusion: 1) the ability to measure TSST-1 in biologic fluids lends stronger support for the role of TSST-1 in menstrual TSS patients; 2) the serologic data support the etiologic role of TSST-1 in menstrual TSS and in nonmenstrual TSS patients from whom toxigenic S. aureus could be cultured, but not for nonmenstrual TSS women from whom toxigenic S. aureus was not isolated; 3) immunoblotting results with acute and convalescent sera from TSS and control patients, not only add further support to the role of TSST-1 in patients from whom toxigenic S. aureus could be isolated, but also indicate that there may be several other staphylococcal products implicated in TSS, particularly in whom antibody to TSST-1 pre-existed in acute sera. The nonresponsiveness or lack of seroconversion to TSST-1 in some patients could suggest either: a) TSST-1 was not the etiologic agent for such patients; b) TSST-1 was the etiologic agent, but the exposure was sufficient for an immune response (similar to tetanus), or; c) some immunologic defect may be present. Future studies are required to clarify these possibilities.
Science, Faculty of
Microbiology and Immunology, Department of
Graduate

Lipopolissacarídeos (LPS), pode tanto proteger quanto exacerbar o desenvolvimento da asma. LPS inicia a ativação da resposta imune via ligação da molécula Toll-like receptor 4 (TLR4) que sinaliza por duas vias distintas, as moléculas adaptadoras MyD88 e TRIF. LPS é um adjuvante que induz resposta do tipo Th1, enquanto que o hidróxido de alumínio (Alum) desperta respostas Th2, porém, a mistura de ambos adjuvantes na indução da resposta alérgica pulmonar ainda não foi investigada. No presente estudo, nós determinamos o efeito de dois agonistas de TLR4, um natural (LPS) e outro sintético (ER-803022) adsorvidos ao Alum sobre o desenvolvimento de doença alérgica pulmonar. Os animais foram sensibilizados pela via subcutânea com os antígenos, Ovoalbumina (OVA) ou Toxóide Tetânico (TT) na presença ou ausência de agonistas de TLR4 co-adsorvidos ao Alum e desafiados com os respectivos antígenos pela via intranasal. Nossos resultados mostraram que a sensibilização com OVA ou TT e LPS coadsorvidos ao Alum, impede o estabelecimento da resposta alérgica mediada por linfócitos Th2, tais como, influxo de eosinófilos, produção de citocinas do tipo 2, hiperreatividade brônquica, secreção de muco, e produção de IgE ou IgG1 anafilática. Apesar dos níveis de IgG2a, isotipo associado com as respostas Th1 estarem aumentados, análise da histopatologia pulmonar não revelou um desvio para o padrão Th1 de inflamação. Verificamos que a presença das moléculas TLR4, MyD88, IL-12/IFN-g mas não TRIF foram necessários para LPS exercer seu efeito inibitório. O agonista sintético de TLR4, menos tóxico que LPS, também protegeu contra o desenvolvimento de inflamação alérgica pulmonar. Em conclusão, nosso trabalho esclarece o efeito da sinalização do TLR4 na sensibilização alérgica e indica que agonista sintético de TLR4 com baixa toxicidade, pode ser utilizado para modular a capacidade adjuvante do Alum e conseqüentemente diminuir a indução de alergias.
Epidemiological and experimental data suggest that bacterial lipopolysaccharides (LPS) can either protect from or exacerbate allergic asthma. LPS triggers immune responses through Toll-like receptor (TLR) 4 that in turn activates two major signaling pathways via either MyD88 or TRIF adaptor proteins. LPS is a pro-Th1 adjuvant while aluminum hydroxide (Alum) is a strong Th2 adjuvant, but the effect of mixing both adjuvants on development of lung allergy has not been investigated. We determined whether natural (LPS) or synthetic (ER-803022) TLR4 agonists adsorbed onto alum adjuvant affect allergen sensitization and development of airway allergic disease. To dissect LPS-induced molecular pathways we used TLR4, MyD88, TRIF, or IL-12/IFN-g deficient mice. Mice were sensitized subcutaneously to allergens such as ovalbumin (OVA) or tetanus toxoid (TT) with or without TLR4 agonists coadsorbed onto Alum and challenged twice via intranasal route with the same allergens. The development of type 2 immunity was evaluated 24 h after last allergen challenge. We found that sensitization with OVA or TT plus LPS co-adsorbed onto Alum impaired allergeninduced Th2-mediated responses such as airway eosinophilia, type 2 cytokines secretion, airway hyperreactivity, mucus hyper production and serum levels of IgE or IgG1 anaphylactic antibodies. Although the levels of IgG2a, a Th1 affiliated isotype increased, investigation into the lung-specific effects revealed that LPS did not induce a Th1 pattern of inflammation. LPS impaired the development of Th2 immunity, signaling via TLR4 and MyD88 molecules via the IL-12/IFN-g axis, but not through TRIF pathway. Moreover, the synthetic TLR4 agonists that proved to have a less systemic inflammatory response than LPS also protected against allergic asthma development. TLR4 agonists co-adsorbed with allergen onto Alum down modulate Th2 immunity and prevent the development of polarized T cell-mediated airway inflammation. Thus, our work clarifies the effect of TLR4 signaling in allergic sensitization and indicates that TLR4 agonists with low toxicity might be useful for down regulating the pro-Th2 adjuvant activity of alum and consequently decrease the induction of allergy.

Le médicament prophylactique qui a drastiquement réduit l’impact et la sévérité du tétanos sur les populations humaines est le vaccin antitétanique. Son principe actif appelé anatoxine tétanique résulte de l’inactivation au formaldéhyde de la toxine tétanique. Cette détoxification chimique est une étape critique qui détermine la sécurité, l’antigénicité et l’immunogénicité du vaccin. Pour une meilleure compréhension de ce processus chimique, à l’échelle moléculaire, nous avons dans un premier temps caractérisé l’anatoxine tétanique par chromatographie liquide couplée à la spectrométrie de masse en tandem haute résolution (LC-MS/MS) afin d’identifier et de localiser exhaustivement l’ensemble des modifications induites par le formaldéhyde sur la structure tridimensionnelle de la protéine vaccinale. Dans un second lieu, pour un meilleur suivi qualité du procédé industriel de fabrication de l’anatoxine tétanique, nous avons développé des méthodes uniques d’expertise in vitro par LC-MS/MS pour réaliser la quantification relative et/ou absolue de la toxine tétanique, de l’anatoxine tétanique, ainsi que pour effectuer la quantification relative des fragments de toxine chimiquement modifiés par le formaldéhyde. Ces outils de caractérisation sont complémentaires aux méthodes de contrôles qualités existantes et contribuent actuellement à un meilleur suivi de la reproductibilité des lots de vaccins antitétaniques
The prophylactic drug, which has drastically reduced the impact and severity of tetanus on human populations, is the tetanus vaccine. Its active ingredient called tetanus toxoid results from the inactivation of tetanus toxin with formaldehyde. This chemical detoxification is a critical step, which determines the safety, antigenicity and immunogenicity of the vaccine. For a better understanding of this chemical process, at the molecular level, we first characterized tetanus toxoid by liquid chromatography coupled with high-resolution tandem mass spectrometry (LC-MS/MS) in order to fully identify and map all the modifications induced by formaldehyde on the three-dimensional structure of the vaccine protein. In a second step, for a better quality control of the industrial process of manufacturing tetanus toxoid, we developed in vitro expertise methods by LC-MS/MS to perform the relative and/or absolute quantification of tetanus toxin, tetanus toxoid, and to carry out the relative quantification of the toxin fragments chemically modified with formaldehyde. These characterization tools are complementary to existing quality control methods and currently contribute to better monitoring the reproducibility of tetanus vaccine batches

O processo de micrencapsulação de proteínas em microesferas (MS) de PLGA [poli (ácido lactico-co-glicolico)] é fácil de fazer e é uma ferramenta útil para melhorar tanto uma formulação quanto para aumentar a atividade imunológica de vacinas de novas gerações. A MS-PLGA têm caráter adjuvante porque é um sistema particulado e, além disto, controla a liberação do antígeno. O escopo desta tese foi o de dar uma nova roupagem para um antígeno vacinal tradicional e muito bem estudado- o toxoide diftérico (Dtxd). Estudaram-se a produção de MS de tamanho desejado; os mecanismos que controlam danos nas proteínas durante o processo de micrencapsulação; a produção de microesferas com características de liberações em tempos distintos e ensaios biológicos. O tamanho de MS é um determinante fundamental para controlar a velocidade de liberação de um soluto. Para se produzir MS com tamanhos controlados usou-se um desenho fatorial experimental com três fatores distintos e três pontos centrais, para se determinar a influência das variáveis (concentração de poli álcool vinílico; velocidade de agitação e relação fase dispersa/fase contínua) na determinação do tamanho das MS. Foram obtidas MS esféricas e lisas de 4- 15 µm de diâmetro. Estes resultados abrem a possibilidade de se formular PLGA-MS com tamanhos planejados através de um mínimo de experimentos. O mecanismo de danos conformacionais nas proteínas nas várias fases do processo de produção de PLGA-MS é ainda uma questão em aberto. Usaram-se várias técnicas biofísicas (HPLC, espectroscopias no uv, fluorescência e CD) além de ELISA para se testar a interferência dos sais da série de Hofmeister sobre a solubilidade e estabilidade da proteína durante a emulsificação e do contacto com a interface água/cloreto de metileno (primeira etapa do processo de preparação de MS). Estudaram-se também a influência de oligômeros de PLGA e SDS sobre a estrutura da proteína no meio de liberação (etapa de liberação do soluto). A emulsificação de Dtxd na presença de Mg2+ induziu agregação protéica, com exposição de resíduos hidrofóbicos para o meio; variações no ângulo diédrico do S-S proteico e perda de identidade imunológica. Esta agregação foi quase abolida pelo caotrópico SCN- (toxicidade = 30 g/ homem adulto de 70 kg). A conformação \"nativa\" do Dtxd e sua atividade biológica foram protegidas pelo KSCN. Os oligômeros de PLGA e o SDS induziram uma conformação de Dtxd nova. A adição de KSCN na fase aquosa aumentou a eficiência de encapsulação de Dtxd pela PLGA-MS em 20 %. Esta foi a solução mais simples quando comparada com aquelas descritas na literatura. Produziram-se seis formulações diferentes (diferentes massas molares e carboximetilações do PLGA) com pelo menos três cinéticas de liberações distintas. Imunizaram-se camundongos com 5 µg de Dtxd encapsulado em MS-PLGA usando-se dois polímeros de 12 kDa (-COOH livre ou metilado) e um outro de 63 kDa (metilado). O padrão de resposta e a maturidade imunológicas foram medidos por titulações de IgG1 e IgG2a. Mantiveram-se os mesmos padrões de resposta humoral (desejável). Menores quantidades de antígenos foram necessárias para se obter os mesmos benefícios gerados pela vacina tradicional de Dtxd. Aumentaram-se a produção e a seletividade de anticorpos através de duas manipulações simples: a formulação e o tempo da aplicação da dose de reforço. Estes resultados colocam estas formulações na área de vacinas de sucesso uma vez que também foram obtidas memórias imunológicas.
The protein microencapsulation within microspheres (MS) of PLGA (Poly-lactide-co-glycolide) is easy to do and, it is a useful tool to enhance formulation and immunologic performances for new generation vaccines. MS-PLGA has adjuvant character because it is a particulate system and can control the antigen release. The question addressed in this thesis was to give this new dress for the traditional and well studied vaccine antigen - the diphtheria toxoid (Dtxd). The steps of MS control size production; mechanism to control protein damages; MS production with different polymers and biological assay were addressed here. MS size is a primary determinant of solute release velocity. A full factorial experimental design 23 with triplicate at the central point was used to determine the influence of variables (polyvinyl alcohol concentration, stirring velocity and the relationship between dispersed /continuous phase) on MS size. Uniformly spherical and smooth microspheres (4 - 15 µm of diameter) were obtained. These results open the possibility of formulating PLGA microspheres with custom sizes performing a minimum of experiments as required for specific applications. It stills an open question to detail the conformational mechanism of protein damages during the various steps of the PLGA microencapsulation process. Various techniques (HPLC gel filtration, ELISA, Fluorescence, UV and Circular dichroism spectroscopies) were tested on the interference of the Hofmeister ion series over protein solubility and stability during the emulsification and contact with the interface water/CH2Cl2 interface (First step on MS preparation). The interference of SDS and PLGA olygomers over protein structure in the liberation media was also studied (solute liberation step). The Dtxd emulsification in the presence of Mg2+ was followed by protein aggregation, with exposition of hydrophobic residues and changes on the dihedral S-S protein angle and loses on immunological identity. This aggregation is 95% avoided by the chaotropic and little toxic salt KSCN (30g/ adult human of 70 kg). All the \"native\" Dtxd conformation and biological properties were maintained by KSCN. MS with different liberation kinetics profile and different erosion characteristics were obtained by using six different polymers. The SDS and PLGA olygomers exerted a generation of new Dtxd molecular organization. The KSCN increased Dtxd encapsulation within PLGA-MS in more than 20 %. This was the simplest solution used to solve protein aggregation compared with others solutions used in the literature. The six different formulations produced (differing in molar mass and carboxymethylation) produced, at least, three different Dtxd liberation profiles. Mice were primed with 5 µg of Dtxd microencapsulated within MS prepared with 12 kDa (ended carboxymethylated or free PLGA) and with 63 kDa (methylated) PLGA. The response patterns and the immune maturity were measured by IgG1 and IgG2a titrations. The humoral pattern was maintained, but fewer antigens were needed to obtain the same traditional Dtxd vaccine benefits. The simple change on Dtxd-PLGA formulation and timing of the booster enhanced both, antibody production and selectivity. An immunological memory was also obtained, putting so, these formulations in the field of successful vaccine.

Repeated administration of a bacterial superantigen, toxic shock syndrome toxin-1 (TSST-1), has been shown to induce CD4+ regulatory T cells in mice. However, it is not clear if the characteristics of TSST-1-induced Tregs differ from those of Tregs induced by other bacterial superantigens, such as staphylococcal enterotoxin A (SEA). The mechanisms of T TSST-1-induced Tregs are also not well characterized. Because in experimental settings Tregs can be used to effectively treat multiple diseases, the potential application of TSST-1-induced Tregs also needs to be determined. In this study, a side-by-side comparative study of Tregs induced by TSST-1 and SEA was conducted. Results showed that TSST-1-induced Tregs were different from those induced by SEA in suppressive function, cytokine profile and proliferative ability. Remarkably, TSST-1-induced Tregs were more potent than SEA-induced Tregs in suppressive activities and proliferative ability in vitro. The possible mechanism of TSST-1-induced Tregs was then investigated. TSST-1-induced Tregs did not induce death of target cells, inhibit the activation of target cells, or cause their target cells to acquire regulatory functions. Supernatants from TSST-1-induced Tregs were not suppressive and blockade of IL-10 by a monoclonal antibody did not reverse the suppression. In contrast, cell contact with target cells was required. In addition, TSST-1-induced Tregs were able to compete with their target cells for IL-2. Finally, the potential therapeutic application of TSST-1-induced Tregs was examined by determining their ability to control acute graft-versus-host disease (aGVHD) in a murine model. Data showed that TSST-1-induced Tregs were able to mediate bystander suppression of aGVHD following re-activation upon administration of TSST-1 post transplant. The Tregs inhibited the production of proinflammatory cytokines triggered by alloresponses, but neither affected the engraftment or expansion of donor T cells nor enhanced the elimination of host APCs. Blockade of IL-10 by in vivo administration of a monoclonal antibody did not reverse the suppression. Finally, although TSST-1-induced Tregs attenuated aGVHD, graft-versus-tumor (GVT) effects were preserved, suggesting that these cells differentially regulate aGVHD versus GVT effects.
Medicine, Faculty of
Medicine, Department of
Experimental Medicine, Division of
Graduate

Blood supply is crucial for tumor growth and metastasis. However, current anti-angiogenic therapy is not as effective as predicted, thus a better understanding of the tumor angiogenic process and new anti-angiogenic agent are urgently required. Anthrax lethal toxin (LeTx) has an anti-angiogenic effect on tumors. Tumors treated with LeTx are smaller, paler, and have lower mean vessel density compared to control treated tumors. Most interestingly, compared to current anti-angiogenic treatment, LeTx does not cause normalization of tumor vessels. Instead, tumors treated with LeTx have massive hemorrhages, pointing to a potential alternative mechanism to inhibit tumor angiogenesis. I hypothesize that instead of causing “normalization” of tumor vasculature, LeTx’s anti-angiogenic effects works in a manner similar to a hemorrhagic toxins. To test this hypothesis, I compared the effect of LeTx to snake venom metalloproteinase, a known hemorrhagic toxin, in tumor vasculature. Quantified by Nuance multispectral imaging system, both LeTx and SVMP caused an increase in tumor hemorrhage. Futher analysis of vasculature integrity using continued vessel length showed disruption of vessels by LeTx and SVMP. With these results, I conclude that the anti-angiogenic effects of LeTx are due to its hemorrhagic nature, and not due to normalization of tumor vasculature. Further understanding of LeTx mechanism can help design novel anti-angiogenic agent that compliments current therapy.

Thesis (M.S.)--University of Missouri-Columbia, 2005.
The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Title from title screen of research.pdf file viewed on (July 13, 2006) Includes bibliographical references.

The report discusses the destruction of toxic and non-toxic solids, liquids and gases through lasers. In order to completely understand the project first chapters describes the basics about laser and plasma separately, from definition to types, components and categories. Differences between laser and microwave system are covered in this chapter as well. Besides lasers there are different technologies that are currently being used to destroy toxic and non-toxic materials. These technologies were studied and comparison tables are made in order to discern between different destruction technologies. For the destruction of toxic and non-toxic materials through lasers two mathematical models have been developed, molecular dissociation model and plasma exploitation model, and later the experimental work was carried out on one of the toxic material. Mathematical modeling and experimental work is in accordance with each other as discussed in results and discussion. Mathematical model shows that all the materials discussed in the report can be destroyed by lasers but in order to carry further experiments on all other toxic and non-toxic materials, a proposal is made for the laser reactor using CAD model (Solid Edge) and drawing software (AutoCAD). Tables and mathematical calculations have been placed in appendix at the end of the report.

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Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq); National Institute of Health (NIH).
O HTLV-1 é o agente etiológico da leucemia/linfoma de células T do adulto (ATLL) e da mielopatia associada ao HTLV-1 (HAM/TSP). Tem-se documentado que células mononucleares de indivíduos infectados não proliferam quando estimuladas com antígenos não relacionados ao vírus como, por exemplo, derivado proteico purificado de Mycobacterium tuberculosis (PPD) e toxoide tetânico (TT). Alguns fatores que podem estar relacionados a essa falta de resposta são as funções de células T regulatórias e disfunção de células apresentadoras de antígeno. Objetivo: Avaliar a resposta imune de indivíduos infectados pelo HTLV-1 ao toxoide tetânico. Materiais e Métodos: Foram selecionados portadores assintomáticos do HTLV-1 baixo produtor de IFN- e controles sadios. Realizou-se sorologia para TT. Os indivíduos soronegativos para TT foram imunizados. Antes e após imunização, fez-se a sorologia para TT e avaliação da expressão de citocinas (IFN- , TNF e IL-10) por linfócitos T CD4+ e T CD8+ estimulados com TT. Os monócitos dos pacientes e controles, estimulados com TT, foram avaliados para a expressão de HLA-DR, CD80, CD86, TNF, IL-12 e IL-10 antes da imunização. Resultados: Após imunização, os pacientes apresentaram menores títulos de IgG anti-TT quando comparados com os controles (p = 0,007). As células mononucleares dos pacientes, estimuladas com TT, não aumentaram a produção de IFN- , TNF e IL-10 após imunização. A frequência de linfócitos T CD4+ expressando IFN- , TNF e IL-10, após estímulo, foi menor nos pacientes do que nos controles pós-imunização (p = 0,01, p = 0,04 e p = 0,01, respectivamente). Os monócitos dos pacientes não aumentaram a expressão de HLA-DR após estímulo com TT. A expressão de TNF e IL-12 por monócitos de pacientes elevaram-se após estímulo com TT (p = 0,009 e p = 0,006, respectivamente). Conclusões: Os indivíduos infectados pelo HTLV-1, após esquema de vacinação, apresentaram diminuição da resposta imune humoral e celular contra TT. Os monócitos destes pacientes exibiram uma disfunção na apresentação antigênica através do mecanismo de expressão de HLA-DR, porém, o segundo sinal (expressão de CD80 e CD86) e expressão de citocinas não apresentaram anormalidades. Tais resultados sugerem que estes mecanismos imunológicos podem participar no aumento da susceptibilidade dos indivíduos infectados pelo HTLV-1 a adquirir outras doenças infecciosas.
Salvador

Traditional remediation techniques in removing toxic metal contaminants using physical and chemical methods are expensive and may cause other forms of damage to the environment, comparing with these techniques bioremediation can serve as an inexpensive, effective and environmental friendly remediation method. This thesis mainly discusses different bioremediation techniques and identifies possible areas in Hong Kong for bioremediation and suggests bioremediation methods for each potential area. Bioremediation of toxic metals is the use of microorganisms, plants, or even larger sized organisms to decontaminate sites with toxic metals. Bioremediation includes phytoremediation, microremediation and vermiremediation which use plants, microorganisms and earthworms to remediate contaminated environments respectively. The 4 most common mechanisms in phytoremediation of toxic metals are phytoextraction, phytofiltration, phytovolatilization and phytostabilization. Phytoremediation are used frequently for remediation around the world and its development includes using well-understood technology and genetic engineering to increase its effectiveness. Microremediation is another promising technology in bioremediation of toxic metals and consists of 6 major mechanisms which are biosorption, bioaccumulation, biotransformation, bioleaching, biomineralization and microbially-enhanced chemisorption of metals. Microremediation is mainly in research phase and its development includes identifying new species, combining with phytoremediation and genetic engineering. Vermiremediation is another rapidly developing technique in bioremediation of toxic metals, assisting other bioremediation by burrowing actions of earthworms and its excretion, and accumulating toxic metals inside their bodies. Vermiremediation is also in research phase but it is rapidly developing. Generally, bioremediation is around 60% cheaper than traditional remediation methods and no pollutants are emitted during the process. However the remediation process is slow and generally takes longer than a year. Sources of toxic metals in contaminated areas in Hong Kong are mainly due to historic industrial discharge although present activities also contribute. Potential areas include sites for electronic waste activities, sediments of Kwun Tong typhoon shelter and sediments of Tolo Harbour.
published_or_final_version
Environmental Management
Master
Master of Science in Environmental Management

Cholera toxin (CT), secreted from Vibrio cholerae, causes a massive fluid and electrolyte efflux in the small intestine that results in life-threatening diarrhea and dehydration which impacts 3-5 million people per year. CT is secreted into the intestinal lumen but acts within the cytosol of intestinal epithelial cells. CT is an AB5 toxin that has a catalytic A1 subunit and a cell binding B subunit. CT moves from the cell surface to the endoplasmic reticulum (ER) by retrograde transport. Much of the toxin is transported to the lysosomes for degradation, but a secondary pool of toxin is diverted to the Golgi apparatus and then to the ER. Here the A1 subunit detaches from the rest of the toxin and enters the cytosol. The disordered conformation of free CTA1 facilitates toxin export to the cytosol by activating a quality control mechanism known as ER-associated degradation. The return to a folded structure in the cytosol allows CTA1 to attain an active conformation for modification of its Gs? target through ADP-ribosylation. This modification locks the protein in an active state which stimulates adenylate cyclase and leads to elevated levels of cAMP. A chloride channel located in the apical enterocyte membrane opens in response to signaling events induced by these elevated cAMP levels. The osmotic movement of water into the intestinal lumen that results from the chloride efflux produces the characteristic profuse watery diarrhea that is seen in intoxicated individuals. The current model of intoxication proposes only one molecule of cytosolic toxin is required to affect host cells, making therapeutic treatment nearly impossible. However, based on emerging evidence, we hypothesize a threshold quantity of toxin must be present within the cytosol of the target cell in order to elicit a cytopathic effect. Using the method of surface plasmon resonance along with toxicity assays, I have, for the first time, directly measured the efficiency of toxin delivery to the cytosol and correlated the levels of cytosolic toxin to toxin activity. I have shown CTA1 delivery from the cell surface to the cytosol is an inefficient process with only 2.3 % of the surface bound CTA1 appearing in the cytosol after 2 hours of intoxication. I have also determined and a cytosolic quantity of more than approximately .05ng of cytosolic CTA1 must be reached in order to elicit a cytopathic effect. Furthermore, CTA1 must be continually delivered from the cell surface to the cytosol in order to overcome the constant proteasome-mediated clearance of cytosolic toxin. When toxin delivery to the cytosol was blocked, this allowed the host cell to de-activate Gs?, lower cAMP levels, and recover from intoxication. Our work thus indicates it is possible to treat cholera even after the onset of disease. These findings challenge the idea of irreversible cellular toxicity and open the possibility of post-intoxication treatment options.
M.S.
Masters
Molecular Biology and Microbiology
Medicine
Biomedical Sciences; Biomedical Sciences

La toxine bactérienne de Shiga se lie au glycosphingolipide (GSL) globotriaosylcéramide (Gb3) afin d’entrer par endocytose dans les cellules en utilisant une voie dépendante et indépendante de la clathrine. Dans la voie indépendante de la clathrine, la toxine de Shiga réorganise les lipides de la membrane de façon à imposer une contrainte mécanique sur la bicouche, conduisant ainsi à la formation de pic d’invagination d'endocytose profonds et étroits. Mécaniquement ce phénomène n’est pas encore compris, notamment il reste énigmatique, comment se traduisent les propriétés géométriques de l’agrégation des glycosphingolipides GSLS et de la toxine. Dans mon travail de thèse, via l’utilisation de la sous-unité B de la toxine de Shiga (STxB) comme un modèle, différentes espèces moléculaires de son récepteur Gb3 ont été synthétisés avec des structures délibérément choisis. Les études réalisées par imagerie de haute résolution et par la modélisation informatique ont permis d’élucider les contraintes mécano-chimique sous-jacente conduisant à une réorganisation efficace qui a pour résultat l’agrégation de la toxine et la réorganisation des lipides. En combinant des expériences de simulation sur ordinateur de dynamique des particules dissipatives (DPD) et des expériences sur des modèles de membranes cellulaires, nous avons fourni la preuve de l’induction d’une force de fluctuation-membrane, de type « force de Casimir », conduisant à l'agrégation des molécules de toxines associées à la membrane à des échelles de longueur mésoscoiques. Nous avons observé et mesuré, en outre la condensation lipidique induite par la toxine, quantitativement sur des monocouches de Langmuir en utilisant la réflectivité des rayons X (XR) et par la mesure de la diffraction des rayons X par incidence rasante (GIXD), fournissant ainsi une preuve directe de l'hypothèse que la toxine a le potentiel de réduire de façon asymétrique la surface moléculaire sur la partie membranaire exoplasmique, ce qui conduit à une déformation locale de la membrane. Durant ma thèse, nos efforts ont été consacrés à la réalisation de nouveaux glycosphinolipides (GSL) comme outils chimiques à visée biologique. Par ailleurs, une nouvelle stratégie de reconstitution de GSL fonctionnels sur la membrane cellulaire, basée sur une réaction de ligation de type « click » entre un glycosyl-cyclooctyne et un azido-sphingosine a été étudiée. Les résultats obtenus sur les cellules se sont avérés beaucoup moins efficace que ceux in vitro. Une poursuite de l'optimisation de cette méthodologie est actuellement en cours. Une sonde fluorescente du glycosphinolipide Gb3, marquée à l’Alexa Fluor 568 lui-même lié par l'intermédiaire d'un bras PEG-α à la position de la chaîne acyle, a été synthétisée. Cette sonde se lie à la STxB sur couche mince de TLC, mais pas sur des membranes modèles. D'autres améliorations sont discutées
Bacterial Shiga toxins bind to the glycosphingolipid (GSL) globotriaosylceramide (Gb3) to enter cells by clathrin-dependent and independent endocytosis. In the clathrin-independent pathway, Shiga toxin reorganizes membrane lipids in a way such as to impose mechanical strain onto the bilayer, thus leading to the formation of deep and narrow endocytic pits. Mechanistically how this occurs is not yet understood, and notably how the geometric properties of toxin-GSLs complexes translate into function has remained enigmatic. In my thesis work, using the B-subunit of Shiga toxin (STxB) as a model, different molecular species of its receptor Gb3 have been synthesized with deliberately chosen structures, coupled with high resolution imaging and computational modeling, to understand the underlying mechano-chemical constraints leading to efficient toxin clustering and lipids reorganization. By combining dissipative particle dynamics (DPD) computer simulation and experiments on cell and model membranes, we provided evidence that a membrane fluctuation-induced force, termed Casimir-like force, drives the aggregation of tightly membrane-associated toxin molecules at mesoscopic length scales. Furthermore, toxin-induced lipid condensation was observed and measured quantitatively on Langmuir monolayers using X-ray reflectivity (XR) and grazing incidence x-ray diffraction (GIXD), thereby providing direct evidence for the hypothesis that the toxin has the potential to asymmetrically reduce the molecular area of the exoplasmic membrane leaflet, leading to local membrane deformation. During my PhD, effort was also invested to develop new GSL tools applied to the biological setting. A novel strategy based on the Cu-free click reaction between glycosyl-cyclooctyne and azido-sphingosine was designed with the goal to functionally incorporate GSLs into cellular membranes. Following the synthesis work, click reactions have been performed in solution and on cells. Compared to the former, results on cells were far less efficient. Further optimization is currently ongoing. A fluorescently labeled Gb3 probe with Alexa Fluor 568 coupled via a PEG linker to the α-position of the acyl chain, was synthesized, to which STxB bound on TLCs, but not on model membranes. Further improvements are discussed

Toxic leadership costs organizations millions at a time in lost employees, lost customers, lost productivity, and even lost health. The literature shows toxic leadership extends beyond just leaders into an interconnected ?toxic triangle? of destructive leaders, conducive environments, and susceptible followers. This study explored, ?Can a free, online mindfulness-based stress reduction course reduce the negative impact of toxic leadership on the organization?? Ten volunteers self-identified as currently working under a toxic leader. The study used an explanatory sequential mixed methods design to measure resistance, compliance, and core self-evaluation along with interviews and journals. The results indicated mindfulness did reduce the negative impact: conducive environments were less conducive and susceptible followers were less susceptible. Additionally, mindfulness had influence on the entire toxic triangle and resulted in unique Toxic Triangle Influence Maps for each situation. Finally, family systems theory was found to be particularly useful for understanding leadership in a toxic triangle.

It was discovered in our laboratory that the bimetallic system palladium on iron (palladized-iron, Pd/Fe) rapidly and completely hydrodechlorinates aqueous solutions of ubiquitous chlorinated aliphatic and aromatic environmental contaminants such as TCE (trichloroethylene), chlorophenols and PCBs (polychlorobiphenyls) at room temperature and atmospheric pressure. When TCE comes in contact with the Pd/Fe surface, it forms ethane as the major gaseous product. Chlorophenols are converted to phenol and PCBs are converted to biphenyl. It was also discovered that through a series of redox reactions metallic iron completely reduces perchlorate to chloride. The objectives of this research are: to show that two iron materials can completely dechlorinate aqueous solutions of toxic chlorinated compounds; to begin to understand how these reactions occur, and; to understand the implications of oxidizing iron. In this research, batch and scaled-up systems were used for fundamental and practical studies of the Pd/Fe system. TCE, chlorophenols and PCBs were the model compounds for these studies. The batch reactions were performed in 1 OmL vials containing 2-5g Pd/Fe and 2-5mL of an aqueous solution of a reactant. The scaled-up reactions were performed in a closed-loop column apparatus with 700g of Pd/Fe and approximately 250mL of an aqueous solution of a reactant. The batch reactions were used to: show complete and rapid hydrodechlorination of the reactants to their products; demonstrate the performance of various types of iron; determine the sequential order of the removal of the chlorine substituents from chlorophenols and PCBs; observe the effect of an HCl-treated and an untreated iron surface prior to palladium deposition and the effects these surfaces have on the hydrodechlorination reactions of chlorophenols and PCBs; and determine what implications iron oxide build-up on the Pd/Fe surface may have on scaled-up Pd/Fe systems. The scaled-up column apparatus was used to determine the longevity of Pd/Fe surfaces and to find a suitable regeneration method. Because Pd/Fe was found to be a suitable reductant for chlorinated organic molecules, chlorine-containing ions such as ClO₄⁻ were also investigated. It was discovered that HCl-treated iron is a suitable reductant for the reduction of the perchlorate ion at room temperature and atmospheric pressure. Palladized-iron and zero-valent iron have been found to be suitable materials for the remediation of many ubiquitous environmental contammants. The hydrodechlorination of chlorinated compounds occurs completely and rapidly with Pd/Fe. Iron effectively reduces perchlorate ions to chloride ions. Both Pd/Fe and zero-valent iron surfaces are relatively easy to prepare, and can be used at room temperature and atmospheric pressure for the dechlorination reactions.

The endopeptidase activity assay developed for measurement of purified botulinum neurotoxin type A (BoNT/A) in clinical therapeutic preparations has been adopted to provide a specific measure of BoNT/A activity in culture supernatants of proteolytic C. botulinum type A. Electrophoretic studies and inhibition of BoNT/A activity by anti-A antibody confirmed the specificity of the assay. The minimum detection limit was 0.2 MLD50/ml indicating the assay as more sensitive than the standard mouse bioassay or any other in vitro assay available to date. Whilst the assay did not exhibit any cross reactions with non-proteolytic (saccharolytic) clostridia, proteolytic C. botulinum types B and F and C. sporogenes showed some cross reactions. The endopeptidase assay was used to investigate physiological aspects of BoNT/A production by proteolytic C. botulinum type A strain NCTC 7272. Growth studies at 15°C, 25°C and 37°C with strain NCTC 7272 demonstrated that the first appearance of BoNT/A (0.1-1.0 MLD50 ml) occurred during mid-late exponential or early stationary phase of growth. Extracellular BoNT/A formation was not proportional to viable count. Slightly more BoNT/A was detected at 25°C than 37° or 15°C. The results of BoNT/A formation by one of the growth curves at 25°C measured by the endopeptidase assay and mouse bioassays were very similar confirming the specificity of the assay. A simple method was developed to lyre the cells so that BoNT/A formation could be subsequently measured in the endopeptidase assay. The data obtained following lysis of cells and measurement of intracellular BoNT/A showed that both intracellular BoNT/A and total BoNT/A formation is not constitutive but are more closely proportional to viable count than extracellular BoNT/A. Release of BoNT/A from cells was not associated with autolysis. The conversion of BoNT/A from the single-chain to dichain form during growth has been measured. The use of the endopeptidase assay has been also exploited to study BoNT/A formation by this strain within the population of cells. There was only a four-fold difference in BoNT/A production by cells of strain NCTC 7272, and further work in this area is warranted. Attempts were made to use MAPs for the production of monoclonal antibodies to SNAP-25 following cleavage by BoNT/E. Whilst the outcome was unsuccessful, the soundness of the principle was demonstrated

Cadmium is a heavy metal which has been associated with a number of pathological diseases. However, despite the known toxicity of this metal, there is inconclusive evidence about its mechanism(s) of action in the cells. The present study was therefore undertaken with the aim of defining the role of oxidative stress, intracellular Ca²⁺ alteration via phospholipase C- Inositol-1,4,5-triphosphate (PLC-IP3) and mitochondrial-cytochrome c dependent pathways in the etiology of cadmium-induced toxicity in three human cell lines: HepG2 (human hepatoma), 1321N1 (human astrocytoma) and HEK 293 (human embryonic kidney) cell lines after 24hrs exposure to 5, 10 and 50 μM cadmium chloride (CdCl₂). The role of the Nrf2-Keap1-ARE pathway in the adaptive response of these cell lines to Cd exposure was examined, and the possible mechanism(s) involved in the protective response of garlic extracts to CdCl₂ were also investigated. Finally, this work examined changes in the proteomic profile of the three human cell lines after Cd exposure in order to develop suitable biomarkers for Cd toxicity. In summary, this study shows that the oxidative stress induced by Cd occurs by different mechanisms depending on cell type and that Ca²⁺ alteration may play an important role in Cd-induced toxicity in HEK 293 cells, while the mitochondrial-cytochrome c dependent pathway is important in Cd toxicity in all three cell lines. Also, the study shows that Nrf2-Keap1-ARE mediated adaptive response to Cd may be activated by PKCδ and that Cd generally alters the metabolism of exposed cells.

Thesis: M. Arch., Massachusetts Institute of Technology, Department of Architecture, 2017.
Cataloged from PDF version of thesis. Page 123 blank.
Includes bibliographical references (pages 121-122).
In an increasingly toxic world where the average person's body contains 29/35 of the toxins listed on the restricted and hazardous substance list, toxicity is unavoidable. This thesis asks how toxins can re-imagined to become active agents in design. Through the negotiation between hard and soft boundaries this work speculates on an architecture of gradients, densities, and velocities to produce temporal spaces of occupation. The year is 2024. Humanity has settled in a condition of toxic urbanism, contained by the toxic wastelands of the periphery. The Anthropocene has wreaked havoc and produced a world of toxins. Early estimates of the exponential destruction caused by our toxic landscapes of production were misled by constantly shifting metrics of toxicity provided by different agencies, bureaus, and offices. Our remediation efforts were too slow, too costly, and failed to produce any agency in the age of toxicity.We continued to produce superfund sites across the country. Landscapes of toxic air, contaminated soil and polluted water became our second nature. As we shifted from one machine age to the next, the continued autonomy provided to production landscapes allowed increasingly more toxic means of production to be developed, this methodology assured there would be no post-toxic future. Within the confines of toxic urbanism, people suited up in protective suits every day. They wore protection more for peace of mind than protection of body. As we destroyed the land, the interior was perfected, continuous halls stocked with machinery created a perfectly sterile environment that defined people's lives, the sprawling mechanized interiors of the no-stop city had finally been realized. We had come a long way. Ever since humanity created the cave fire, toxins had been part of our environment. The hearth, originally acted as both an object of environment and an object of culture. As we followed the flames into modernism we found ourselves in a state of homelessness explicated by the dichotomy between our technological culture and its toxic means of production. Heidegger, described the sensation as Heimatlosigkeit, the signification of our existential orientation in the era of Gestell. Humanity has however always been a risk adverse society, and as they began to reject the sterile environments of safety for toxic environments of experience agency was produced in the design of toxins. In an increasingly toxic world, this thesis explores how toxins can become active participants and drivers for the production of temporal spaces defined by the hard and soft boundaries they operate within. Architectural interests in materiality and dimension are replaced in favor of velocities, gradients, and densities that define zones of occupiability.
by Maxwell A. Jarosz.
M. Arch.

Online gaming is the entertainment of the masses nowadays as it entertains hundreds of millions of players around the world on daily basis. Along with the popularity of online gaming, phenomenon of toxic behavior has also taken root in those games. Toxic behavior is strongly present in current day online games and very few people are spared from it nowadays. The purpose of this study was to take a closer look into the toxic behavior and how it affects those who actively play online games. This study started by conducting literature review to identify the various different aspects that surround the online gaming in addition to toxic behavior. Different types of toxic behavior were also identified from current literature. The themes that emerged from the literature review formed a backbone for constructing a qualitative interview, the designated data gathering method of this study. Aim of the research interviews was to gather information on how the various different toxic behaviors affect the online gaming of the interviewees who were all active online game players. Data gathered by the interviews was analyzed by conducting inductive content analysis, where prominent themes were brought up for closer inspection and reported in the findings. On a general level, the results of the current study suggested that toxic behavior affects online gaming in a negative way, with very few exceptions. For example, cheating was seen as a behavior that destroys the competition aspect of the game and ruins the game for all the participants, while flaming was often simply discarded as provocation and simply ignored. Tangible toxic behaviors like cheating and griefing were found to affect the gaming experience and flow sensation found in gaming in a very negative way. Unchecked cheating was even found to be a cause for quitting a game for good for some people. The current study found hints of more organized forms of scamming taking place in online games instead of just act that takes place between two players. Even cyberbullying was reported, as multiple stories of players ganging up on single player of lower skill were heard. The current study provides the reader with up-to-date information on toxic behavior in online games and how it affects the online gaming, mainly from the viewpoint of veteran players. Additionally, based on the interview data and the content analysis, a three-tier list on how to fight toxic behavior was gathered for the game developers. Based on the results of the current study some future research actions were suggested. Cyberbullying in online games was found to be one area in need of a more in depth look, and the link between toxic behaviour and flow in online games was found to require further studies.

Isoniazid (INH), one of the most effective agents in the treatment and prophylaxis of tuberculosis associated with a mild increase in liver transaminases in up to 20% of treated patients and severe hepatotoxicity in up to 2% of treated patients. Since the 1970's, a number of studies have been reported on the mechanism of INH-induced hepatotoxicity. The original studies in rats suggested that the hepatotoxic effect of INH was due to the microsomal metabolism of acetylhydrazine (AcHz, a metabolite of INH) to a reactive intermediate. More recently other workers have been unable to reproduce the original results in the rat, or other mechanistic models have been proposed. Therefore, it is necessary to establish a reproducible animal model which resembles INH hepatotoxicity in the human being. In these experiments,, male New Zealand white rabbits were used, and divided into 12 treatment groups of 6-8 rabbits each. Serum argininosuccinate lyase (ASAL) levels and histological changes in liver slides were chosen as indices of hepatotoxicity. After the determination of acetylator phenotype for each rabbit, INH and its metabolites, acetylisoniazid (AcINH), hydrazine (Hz) and AcHz were administered in a two-day regimen orally or subcutaneously. The results showed that the serum ASAL level in rabbits is a sensitive and specific enzyme marker which parallels the incidence of hepatic necrosis seen on histology. The serum ASAL control values 4.3±2.6 (SD) Takahara units were maintained until about 24 hrs after the first challenge of INH in the two-day regimen (0.36+3x0.26 mmol/kg/dx2d, s.c.); peak values of up to 2674 Takahara units occurred at about 72 hrs. No significant difference between the toxicity of INH given orally and subcutaneously was detected. Phenobarbital (PB) (0.1 mg/kgx3d, i.p.) pretreatment increased the elevation of serum ASAL level caused by INH (0.36+3x0.26 mmol/kg/dx2d, p.o.) significantly (p<0.05, F test) compared with the group without PB pretreatment. The 65 experimental rabbits were classified into populations of acetylator phenotype by measuring their acetylation rate of sulfamethazine (SMZ): fast acetylators with a SMZ t[formula omitted] of 12.8±4.4 (SD) (n=54) and slow acetylators with a SMZ t[formula omitted] of 50.3±10.4 (SD) (n=ll). Among the rabbits challenged with INH (0.36+3x0.26 mmol/kg/dx2d, p.o. or s.c.) with or without PB pretreatment, no correlation was found between the peak serum ASAL values and acetylation rate represented by SMZ t[formula omitted (r=0.05, n=18). This lack of this correlation was also present in rabbits challenged with AcINH and Hz. Among INH and its metabolites, AcINH, Hz and AcHz, Hz is the most potent hepatotoxin. Its effect is dose-dependent over the dose range (0.10+3x0.07, 0.14+3x0.10 and 0.19+3x0.14 mmol/kg/dx2d, p.o.). AcHz (0.36+3x0.26 mmol/kg/dx2d, s.c.), produced no significant hepatotoxic effect, which is contradictory to the results reported by other authors. AcINH (0.28+3x0.20 mmol/kg/dx2d, s.c, 0.42+3x0.30 mmol/kg/dx2d, s.c. or p.o.) had a intermediate hepatotoxic effect which is similar to that of INH. The results showed that (1) the rabbit is a reproducible animal model for studying INH hepatotoxicity; (2) the release of ASAL to serum and pathological changes resemble that seen in human beings; (3) the hepatotoxicity of INH is potentiated by PB pretreatment which is in accordance with the evidence in human beings of an increased risk in the presence of microsomal enzyme inducers; (4) the acetylation rate does not affect the hepatotoxicity of INH; (5) among the metabolites tested, Hz was most potent. These data indicate that the hypothesis that INH hepatotoxicity is due to microsomal metabolism of AcHz is probably incorrect. We think that it is more likely that Hz is responsible. Further studies are required to elucidate the exact mechanism in the rabbit model.
Medicine, Faculty of
Anesthesiology, Pharmacology and Therapeutics, Department of
Graduate

Staphylococcus aureus is a gram positive bacterium asymptomatically colonizing 30-40% of the human population. S. aureus causes a variety of infections including superficial skin lesions, toxic shock syndrome, and infective endocarditis (IE). There are 100,000 cases of IE each year in the United States. IE is a life threatening infection of native/prosthetic valves and the lining of the heart. It is characterized by the formation of vegetations, “cauliflower-like” structures composed of bacteria and host factors. S. aureus is the most commonly identified pathogen (up to 40%) in patients with IE. USA200 (Clonal Complex 30) strains of S. aureus are significantly associated with IE, all of which produce toxic shock syndrome toxin-1 (TSST-1) and β-toxin. TSST-1 characterizes the staphylococcal Group I superantigens (SAgs). The major mechanism of activity of TSST-1 and other SAgs is the ability to activate T-cells and APCs by non-specifically cross-bridging Vβ-chains of T-cell receptors (TCRs) with α and/or β-chains of major histocompatibility complex II (MHCII) molecules on antigen presenting cells (APCs). In a rabbit model of IE and sepsis, TSST-1 is critical for the development of vegetations and the associated colony forming units (CFUs). β-toxin has a molecular mass of 35 kDa, a basic pI (>10.0), and is a member of the DNase I superfamily. This cytotoxin has two distinct mechanisms of action: sphingomyelinase (SMase) activity and DNA biofilm ligase activity. β-toxin is critical for causing IE in a rabbit model that strongly resembles human disease. This toxin association had been observed, but studies have not been completed to determine what role TSST-1 and β-toxin play independently and in cooperation with one another, and more specifically which mechanism each uses, during IE infections. While TSST-1 and β-toxin are both important for IE, they are very different toxins. My studies determined that the presence of TSST-1 and β-toxin in combination results in the highest levels of lethality in a rabbit model of IE. A strain expressing TSST-1 lacking superantigenic activity has decreased lethality compared to the same strain expressing wild type TSST-1. My study is the first to begin characterization of the DNA biofilm ligase active site by identifying important residues via a DNA binding and biofilm formation assays. Furthermore, my research shows that a β-toxin mutant lacking SMase activity is decreased in lethality and vegetation formation compared to wild type. β-toxin mutants disrupted in biofilm ligase activity do not decrease lethality but are deficient in vegetation formation compared to wild type. Utilizing in vitro assays to assess cellular events during IE, I established that β-toxin causes changes to morphology and is cytotoxic to human aortic endothelial cells (HAECs), inhibits production of IL-8, and modulates the expression levels of cluster of differentiation 40 (CD40) and vascular cell adhesion molecule 1 (VCAM-1). My work shows these two virulence factors (TSST-1 and β-toxin) produced by USA200 strains and other clonal groups play important roles in causing IE.

Botulinum toxin (BTX) is used in various fields of medicine, including the treatment of hyperhidrosis and cervical dystonia. Botox®, Dysport®, Xeomin® and NeuroBloc® are commercially available BTX products, which are formulated differently and their dosing units are unique. Dosage and concentration of the prepared solution for injection varies considerably among studies comparing the products. Improved guidelines on concentration and dosing when changing from one product to another are warranted. This would ensure the use of the lowest effective doses for good effect, minimal risk of antibody formation and side-effects as well as reduced costs. The aim of the present work was to find the most appropriate BTX concentration for each of the four products to achieve the highest sweat reducing effect and to investigate dose conversion ratios between Botox and Dysport in the treatment of cervical dystonia when the products are diluted to the same concentration, 100 U/ml. Paper I and II clearly confirm that it is crucial to consider the BTX concentration in a treatment regimen, especially when changing between different products. The optimal concentration to reduce sweating varies among the products and was found to be 25 U/ml for Botox and Xeomin, approximately 100 U/ml for Dysport and 50 U/ml for NeuroBloc. However, for NeuroBloc the optimal concentration might be even lower. In Paper III, which is a retrospective study using casebook notes from 75 patients with cervical dystonia, it was found that the most appropriate dose conversion ratio to use when switching from Botox to Dysport was 1:1.7. In Paper IV, Botox and Dysport were prospectively compared in a double-blind, randomized clinical trial in two different dose conversion ratios (1:3 and 1:1.7) when diluted to the same concentration (100 U/ml). No statistically significant difference was seen between Botox (1:3) and Dysport nor between Botox (1:1.7) and Dysport four weeks after treatment. Some of the secondary outcome observations, however, did indicate that the ratio 1:3 resulted in suboptimal efficacy of Botox but this must be further validated in a larger patient material.

Freshwater cyanobacteria produce a wealth of biologically active metabolites, which can adversely affect human and animal health, and cause great economic damage to the fishing, tourism and water-management industries on a global scale. We describe the molecular genetics and biochemistry of biosynthesis for the cyanobacterial toxic alkaloids cylindrospennopsin, paralytic shellfish toxins (PST) and anatoxin-a. Characterisation of the 43 kb cylindrospennopsin biosynthesis gene cluster (cyr), in Cylindrospermopsis raciborskii AWT205 is described. Biosynthesis is initiated via an amidinotransfer onto glycine followed by five polyketide extensions. Rings are formed via Michael additions, while the uracil ring is formed by a novel mechanism. Tailoring reactions, including sulfation and hydroxylation complete the biosynthesis. We describe the characterisation of PST biosynthesis gene clusters in Anabaena circinalis, Aphanizomenon sp. and Lyngbya wollei. These gene clusters span between 28 and 36 kb and contain genes coding for the biosynthesis and export of PSTs. The Lyngbya wollei PST gene cluster represents a 'natural combinatorial biosynthesis' event, explaining its unique toxin profile. A biosynthetic pathway leading to the formation of saxitoxin and its analogues in these organisms is proposed, and a putative insertion/excision site of the PST gene cluster in Anabaena circinalis 310F was identified. Interestingly, PSTs are produced by distantly related organisms via this unique biosynthesis pathway. We Investigated the phylogenetics of PST biosynthesis genes from four different genera of cyanobacteria. The results suggested that PST biosynthesis in cyanobacteria is an ancient trait, whereby the sporadic distribution of PST production in extant isolates of Anabaena circinalis and Aphanizomenon sp. is a result of the repeated loss of the biosynthetic gene cluster. Horizontal gene ransfer also appears to have had a critical influence on PST biosynthesis in Lyngbya wollei. We additionally propose a hypothetical, mixed non-ribosomal peptide synthetase (NRPS)/polyketide synthase (PKS) biosynthesis scheme for anatoxin-a. Degenerate PCR primers were developed, for the specific amplification of mixed NRPSIPKS hybrid ketosynthase (KS) domains. Gene-walking distally to a novel hybrid KS domain in the anatoxin-a producer Planktothrix rubescens, revealed an orphan gene cluster, denoted pro, which spans 24 kb and codes for a mixed NRPS/PKS system, putatively producing an acetylated and sulphated dipeptide.

Thesis (Ph.D.)--University of Cincinnati, 2007.
Advisor: Alison A. Weiss. Title from electronic thesis title page (viewed Feb. 5, 2008). Keywords: Bacillus anthracis, anthrax toxin, AVA. Includes abstract. Includes bibliographical references.