Thesis (M. Med. Sc.)--University of Hong Kong, 2000. / Includes bibliographical references (leaves 36-45).

Thesis (M.S.)--University of Wisconsin--Madison, 1984. / Typescript. Includes tables. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 116-120).

Thesis (M.Med.Sc.)--University of Hong Kong, 2000. / Includes bibliographical references (leaves 36-45). Also available in print.

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

The mechanisms of idiosyncratic drug reactions (IDRs) are unknown; however, most appear to be immune-mediated. Their idiosyncratic nature and the paucity of animal models make mechanistic studies very difficult. One of the few animal models is penicillamine-induced autoimmunity in Brown Norway rats. The major focus of this thesis was the use of this model to study the interaction between penicillamine and macrophages, the involvement of Th17 cells, and extension of this model to idiosyncratic drug-induced liver injury. One of the costimulatory signals leading to T cell activation appears to be reversible Schiff-base formation between an amine on T cells and an aldehyde on macrophages. We hypothesized that penicillamine binds to these aldehydes leading to macrophage activation and autoimmunity. By using biotinylated aldehyde-reactive agents such as ARP, we demonstrated the existence of aldehydes on the surface of macrophages. We synthesized biotinylated-penicillamine and it also binds to macrophages. Several proteins to which ARP binds were identified providing clues to the signal transduction pathways leading to macrophage activation. Biological consequences of this binding were investigated with a microarray study. ARP binding was also observed in the macrophage cell line, RAW264.7, and incubation with penicillamine stimulated the production of TNF-α, IL-6, and IL-23. Hydralazine and isoniazid, which are known to cause a lupus-like syndrome in humans and irreversibly bind to aldehyde groups, were also found to activate RAW264.7 cells. Th17 cells are prominent in autoimmune syndromes and Th17-associated cytokines such as IL-17 were elevated in the penicillamine-treated animals that developed autoimmunity. We have hypothesized that some drug-induced liver injury has an autoimmune component. A pilot study quantified serum concentrations of 26 cytokines/chemokines in patients with various forms of acute liver failure (ALF): idiosyncratic drug-induced ALF, acetaminophen-induced ALF, and viral hepatitis. IL-17 was elevated in 60% of patients with idiosyncratic drug-induced ALF, which supports an autoimmune component in these patients; however, it was also elevated in many cases of acetaminophen-induced ALF, presumably released by the innate immune system. These studies provide important insights into the mechanism of penicillamine-, hydralazine-, and isoniazid-induced autoimmunity and also provide clues to other IDRs that may have an autoimmune component.

The mechanisms of idiosyncratic drug reactions (IDRs) are unknown; however, most appear to be immune-mediated. Their idiosyncratic nature and the paucity of animal models make mechanistic studies very difficult. One of the few animal models is penicillamine-induced autoimmunity in Brown Norway rats. The major focus of this thesis was the use of this model to study the interaction between penicillamine and macrophages, the involvement of Th17 cells, and extension of this model to idiosyncratic drug-induced liver injury. One of the costimulatory signals leading to T cell activation appears to be reversible Schiff-base formation between an amine on T cells and an aldehyde on macrophages. We hypothesized that penicillamine binds to these aldehydes leading to macrophage activation and autoimmunity. By using biotinylated aldehyde-reactive agents such as ARP, we demonstrated the existence of aldehydes on the surface of macrophages. We synthesized biotinylated-penicillamine and it also binds to macrophages. Several proteins to which ARP binds were identified providing clues to the signal transduction pathways leading to macrophage activation. Biological consequences of this binding were investigated with a microarray study. ARP binding was also observed in the macrophage cell line, RAW264.7, and incubation with penicillamine stimulated the production of TNF-α, IL-6, and IL-23. Hydralazine and isoniazid, which are known to cause a lupus-like syndrome in humans and irreversibly bind to aldehyde groups, were also found to activate RAW264.7 cells. Th17 cells are prominent in autoimmune syndromes and Th17-associated cytokines such as IL-17 were elevated in the penicillamine-treated animals that developed autoimmunity. We have hypothesized that some drug-induced liver injury has an autoimmune component. A pilot study quantified serum concentrations of 26 cytokines/chemokines in patients with various forms of acute liver failure (ALF): idiosyncratic drug-induced ALF, acetaminophen-induced ALF, and viral hepatitis. IL-17 was elevated in 60% of patients with idiosyncratic drug-induced ALF, which supports an autoimmune component in these patients; however, it was also elevated in many cases of acetaminophen-induced ALF, presumably released by the innate immune system. These studies provide important insights into the mechanism of penicillamine-, hydralazine-, and isoniazid-induced autoimmunity and also provide clues to other IDRs that may have an autoimmune component.

A Doença de Wilson (DW) é uma moléstia hereditária, caracterizada pela deficiência de excreção do cobre pelo fígado devido à mutação do gene A TP7B. O distúrbio do olfato ocorre com frequência em doenças neurodegenerativas como na doença de Parkinson (DP) e na doença de Alzheimer (DA). A análise do olfato tem sido utilizada como um instrumento útil no diagnóstico diferencial das diversas formas de parkinsonismo degenerativo, e, especialmente, na diferenciação entre DP e tremor essencial. O diagnóstico precoce na DW é a chave para o sucesso do tratamento. Na hipótese de haver comprometimento do olfato em fases iniciais da doença, esse poderia ser um dado a mais para auxiliar no diagnóstico. Até o presente, há apenas um estudo relacionando a DW com a disfunção do olfato. O objetivo deste estudo foi avaliar o olfato em um grupo de pacientes com DW e confrontar com grupo- controle. No presente estudo, foram analisados 37 portadores de DW com manifestação neurológica, 24 portadores de DW sem manifestação neurológica e 59 controles. Todos os indivíduos foram analisados com relação à idade, ao gênero, ao grau de escolaridade, ao uso de tabaco e ao miniexame do estado mental (MEEM), e os portadores de DW foram avaliados quanto ao tempo de doença, tratamento medicamentoso e escore neurológico. O olfato foi avaliado por meio do teste de identificação de odor 8niffin\' 8ticks (88-16 canetas numeradas e quatro opções de resposta para cada uma). Vinte e quatro indivíduos eram pacientes da DW sem manifestação neurológica (45,83% do gênero feminino) e 37 pacientes apresentavam manifestações neurológicas (56,76% do gênero masculino). O qrupo-controle foi composto por 59 indivíduos, 35 (59,33%) do gênero masculino. As médias de- idade foram de 33,38 ± 9,79 anos no grupo de portadores de DW com manifestação neurológica; 29 ± 9,61 anos no grupo de portadores de DW sem manifestação neurológica e 33,81 ± 10,67 anos no grupo-controle. Todos os pacientes com DW estavam em tratamento: 47(77%) com penicilamina, 7 (11,5%) com trientine e 7 (11,5%) com sais de zinco. As médias de respostas corretas no teste de identificação do odor 88-16 foram: 12,03 ± 2, 21 no grupo de portadores de DW com manifestação neurológica, 12, 15 ± 2,07 no grupo de portadores de DW com manifestação hepática e 12,70 ± 2,03 para o grupo- controle. Na avaliação objetiva do olfato com o teste de identificação do odor SS-16, não foi evidenciada diferença significativa entre os três grupos analisados, mas observou-se que o MEEM e o grau de escolaridade influenciaram significativamente no escore do 88-16 na comparação do grupo de pacientes com DW com manifestação neurológica com os outros dois grupos (grupo-controle e o grupo de portadores de DW com manifestação hepática). No presente estudo, não foi evidenciada disfunção olfatória nos pacientes com DW, mas foi observada diminuição da percepção do olfato em alguns pacientes com DW (com e sem manifestação neurológica). Em relação à disfunção olfatória evidenciada em alguns pacientes com DW na presente análise, algumas considerações são pertinentes e poderiam ter influenciado na identificação do olfato neste grupo de pacientes com DW. O acúmulo de cobre e a produção de radicais livres no sistema nervoso central (SNC) podem desencadear processos de neurodegeneração em estruturas envolvidas no olfato, alterações metabólicas, acúmulo de substâncias neurotóxicas (amônia e manganês) e alterações de neurotransmissores, e contribuir para o surgimento da disfunção olfatória / Wilson\'s disease (WO) is a hereditary disease due to a mutation in ATP7B gene, characterized by deficiency of copper excretion by the liver. Smell disorders are frequently encountered in neurodegenerative diseases, such as Parkinson\'s disease (PO) and Alzheimer\'s disease (AO). Smell analysis has been a useful tool for the differential diagnosis of several forms of degenerative parkinsonism, and especially for the differentiation between PO and essential tremor. Early diagnosis in WO is the key for a successful treatment. If there were smell impairment in the early stages of the illness, it could be used as another clue to help on its diagnosis. To the present date, there is only one study connecting WO with smell problems, the aim of this study was to evaluate smell function in a group of WO patients and compare them with a control group. We analyzed 37 WO patients with and 24 WO patients without neurologic symptoms, and 59 controls. Ali subjects were evaluated regarding age, gender, schooling, tobacco use, Mini Mental State Examination (MMSE), and the WO patients were also evaluated regarding duration of the illness, medication and neurologic scoring. Smell was analyzed by means of Sniffin\' Sticks smell identification test (SS-16 numbered pens and four options of answer for each pen). Twenty-four subjects with WO had no neurologic symptoms (45.83% female), and 37 patients had neurologic impairment (56,76% male). The control group was composed by 59 individuais, 35 (59,33%) male. Their age average were 33,38 ± 9,79 years for WO neurologic symptoms; 29 ± 9,61 years for WO without neurologic symptoms; and 33,81 ± 10,67 years for the control group. Ali WO patients were on treatment: 47(77%) with penicillamine, 7(11,5%) with trientine, and 7(11,5%) with zinc salt formulations. The average of correct answers in the SS-16 were: 12,03 ± 2,21 for the WO with neurologic symptoms group; 12,15 ± 2,07 for the WO without neurologic symptoms; and 12,70 ± 2,03 for the control group. In the smell testing with SS-16, there was no significant difference among the three groups, but the MMSE scoring and schooling had a significant influence over SS-16 score when comparing WO with neurologic symptoms patients with the other groups (WO patients without neurologic symptoms and control group). There was no smell dysfunction in WO patients in this study, but diminished smell perception was observed in some WO patients (either with or without neurologic impairment). Regarding smell impairment observed in some WO patients in the current analysis, some considerations must be made that could have influenced smell identification in these individuais. Copper accumulation and free radicais production in the central neNOUS system can trigger neurodegeneration processes in structures involved in srnell perception, metabolic impairment, building up of neurotoxic substances (such as ammonia and manganese), and neurotransmitter disorders, contributing to the emergence of srnell dysfunction

The significantly increased copper and oxidative stress levels are characteristic hallmarks of cancer cells. These differences provide a unique opportunity for selective targeting of cancer cells. D-penicillamine (D-pen) has been proposed to generate reactive oxygen species (ROS) in presence of copper. Therefore, these studies were aimed at investigating the potential application of a currently marketed copper chelator, D-pen, as a novel cytotoxic anti-cancer agent. D-pen was shown to produce ROS, specifically hydrogen peroxide (H2O2), in the presence of cupric sulfate through a copper catalyzed oxidation reaction. During this process D-pen was converted to D-pen disulfide. The experimental proof of the H2O2 generation was conclusively shown with the aid of a novel High Performance Liquid Chromatography (HPLC) assay. The in-vitro cytotoxicity of D-pen co-incubated with cupric sulfate was examined in human beast cancer (MCF-7 and BT474) and leukemia cells (HL-60, HL-60/VCR, and HL-60/ADR). D-pen was shown to cause concentration dependent cytotoxicity in both leukemia and breast cancer cells. A direct correlation between the detection of intracellular ROS and cytotoxicity was established. The treatment of D-pen plus cupric sulfate resulted in a significant reduction in the intracellular thiol content. D-pen is highly hydrophilic and is rapidly eliminated from the body; therefore to improve the intracellular uptake and to protect the thiol group of D-pen, we carried out the synthesis and the in-vitro characterization of a novel gelatin-D-pen conjugate. It was shown that D-pen alone does not enter cells. Confocal microscopy was employed to exhibit the uptake of the novel gelatin-D-pen conjugate by cancer cells. As the cancer cells in-vitro do not accumulate the same levels of copper as reported for cancer cells in-vivo, cancer cells were pre-treated with cupric sulfate to simulate the elevated copper levels. The cupric sulfate pretreatment resulted in reduced thiol level and significantly increased cellular copper content compared to untreated cells. Whereas both free D-pen and gelatin-D-pen conjugate lacked cytotoxicity in un-treated cells, both agents caused concentration dependent cytotoxicity in cupric sulfate pre-treated leukemia cells. Therefore, it was shown that the administration of D-pen as polymer conjugate would potentially provide cytotoxicity and specificity in the treatment of cancer.

A Doença de Wilson (DW) é uma moléstia hereditária, caracterizada pela deficiência de excreção do cobre pelo fígado devido à mutação do gene A TP7B. O distúrbio do olfato ocorre com frequência em doenças neurodegenerativas como na doença de Parkinson (DP) e na doença de Alzheimer (DA). A análise do olfato tem sido utilizada como um instrumento útil no diagnóstico diferencial das diversas formas de parkinsonismo degenerativo, e, especialmente, na diferenciação entre DP e tremor essencial. O diagnóstico precoce na DW é a chave para o sucesso do tratamento. Na hipótese de haver comprometimento do olfato em fases iniciais da doença, esse poderia ser um dado a mais para auxiliar no diagnóstico. Até o presente, há apenas um estudo relacionando a DW com a disfunção do olfato. O objetivo deste estudo foi avaliar o olfato em um grupo de pacientes com DW e confrontar com grupo- controle. No presente estudo, foram analisados 37 portadores de DW com manifestação neurológica, 24 portadores de DW sem manifestação neurológica e 59 controles. Todos os indivíduos foram analisados com relação à idade, ao gênero, ao grau de escolaridade, ao uso de tabaco e ao miniexame do estado mental (MEEM), e os portadores de DW foram avaliados quanto ao tempo de doença, tratamento medicamentoso e escore neurológico. O olfato foi avaliado por meio do teste de identificação de odor 8niffin\' 8ticks (88-16 canetas numeradas e quatro opções de resposta para cada uma). Vinte e quatro indivíduos eram pacientes da DW sem manifestação neurológica (45,83% do gênero feminino) e 37 pacientes apresentavam manifestações neurológicas (56,76% do gênero masculino). O qrupo-controle foi composto por 59 indivíduos, 35 (59,33%) do gênero masculino. As médias de- idade foram de 33,38 ± 9,79 anos no grupo de portadores de DW com manifestação neurológica; 29 ± 9,61 anos no grupo de portadores de DW sem manifestação neurológica e 33,81 ± 10,67 anos no grupo-controle. Todos os pacientes com DW estavam em tratamento: 47(77%) com penicilamina, 7 (11,5%) com trientine e 7 (11,5%) com sais de zinco. As médias de respostas corretas no teste de identificação do odor 88-16 foram: 12,03 ± 2, 21 no grupo de portadores de DW com manifestação neurológica, 12, 15 ± 2,07 no grupo de portadores de DW com manifestação hepática e 12,70 ± 2,03 para o grupo- controle. Na avaliação objetiva do olfato com o teste de identificação do odor SS-16, não foi evidenciada diferença significativa entre os três grupos analisados, mas observou-se que o MEEM e o grau de escolaridade influenciaram significativamente no escore do 88-16 na comparação do grupo de pacientes com DW com manifestação neurológica com os outros dois grupos (grupo-controle e o grupo de portadores de DW com manifestação hepática). No presente estudo, não foi evidenciada disfunção olfatória nos pacientes com DW, mas foi observada diminuição da percepção do olfato em alguns pacientes com DW (com e sem manifestação neurológica). Em relação à disfunção olfatória evidenciada em alguns pacientes com DW na presente análise, algumas considerações são pertinentes e poderiam ter influenciado na identificação do olfato neste grupo de pacientes com DW. O acúmulo de cobre e a produção de radicais livres no sistema nervoso central (SNC) podem desencadear processos de neurodegeneração em estruturas envolvidas no olfato, alterações metabólicas, acúmulo de substâncias neurotóxicas (amônia e manganês) e alterações de neurotransmissores, e contribuir para o surgimento da disfunção olfatória / Wilson\'s disease (WO) is a hereditary disease due to a mutation in ATP7B gene, characterized by deficiency of copper excretion by the liver. Smell disorders are frequently encountered in neurodegenerative diseases, such as Parkinson\'s disease (PO) and Alzheimer\'s disease (AO). Smell analysis has been a useful tool for the differential diagnosis of several forms of degenerative parkinsonism, and especially for the differentiation between PO and essential tremor. Early diagnosis in WO is the key for a successful treatment. If there were smell impairment in the early stages of the illness, it could be used as another clue to help on its diagnosis. To the present date, there is only one study connecting WO with smell problems, the aim of this study was to evaluate smell function in a group of WO patients and compare them with a control group. We analyzed 37 WO patients with and 24 WO patients without neurologic symptoms, and 59 controls. Ali subjects were evaluated regarding age, gender, schooling, tobacco use, Mini Mental State Examination (MMSE), and the WO patients were also evaluated regarding duration of the illness, medication and neurologic scoring. Smell was analyzed by means of Sniffin\' Sticks smell identification test (SS-16 numbered pens and four options of answer for each pen). Twenty-four subjects with WO had no neurologic symptoms (45.83% female), and 37 patients had neurologic impairment (56,76% male). The control group was composed by 59 individuais, 35 (59,33%) male. Their age average were 33,38 ± 9,79 years for WO neurologic symptoms; 29 ± 9,61 years for WO without neurologic symptoms; and 33,81 ± 10,67 years for the control group. Ali WO patients were on treatment: 47(77%) with penicillamine, 7(11,5%) with trientine, and 7(11,5%) with zinc salt formulations. The average of correct answers in the SS-16 were: 12,03 ± 2,21 for the WO with neurologic symptoms group; 12,15 ± 2,07 for the WO without neurologic symptoms; and 12,70 ± 2,03 for the control group. In the smell testing with SS-16, there was no significant difference among the three groups, but the MMSE scoring and schooling had a significant influence over SS-16 score when comparing WO with neurologic symptoms patients with the other groups (WO patients without neurologic symptoms and control group). There was no smell dysfunction in WO patients in this study, but diminished smell perception was observed in some WO patients (either with or without neurologic impairment). Regarding smell impairment observed in some WO patients in the current analysis, some considerations must be made that could have influenced smell identification in these individuais. Copper accumulation and free radicais production in the central neNOUS system can trigger neurodegeneration processes in structures involved in srnell perception, metabolic impairment, building up of neurotoxic substances (such as ammonia and manganese), and neurotransmitter disorders, contributing to the emergence of srnell dysfunction

A doença de Wilson (DW) é distúrbio da excreção biliar de cobre, de herança autossômica recessiva, devido a mutações no gene ATP7B. O cobre que não se liga à apoceruloplasmina circula no organismo ligado a aminoácidos, deposita-se principalmente no fígado e no cérebro e é excretado pelos rins. A cuprúria maior que 100ug/24h pode auxiliar no diagnóstico, embora cerca 20% dos pacientes com DW apresentem níveis anormais de cuprúria basal. A administração da d-penicilamina (DPA) pode promover, em crianças, valores maiores que 1.600ug/24h. A fim de se conhecer os níveis de cuprúria de possíveis indivíduos heterozigotos adultos, foram avaliados 25 pais e 25 mães de pacientes (média 61 anos em homens; 57 anos em mulheres) com diagnóstico de DW. Foram obtidos os níveis séricos de enzimas hepáticas, cobre e ceruloplasmina, e quantificada a cuprúria de 24h. A seguir, os indivíduos receberam DPA 1,0g por via oral dividido em duas tomadas, durante nova coleta de urina para dosagem de cuprúria de 24h. Esta análise foi realizada pelo método de espectrometria de absorção atômica eletrotérmica. Os níveis de enzimas hepáticas foram semelhantes nos dois grupos, exceto o nível médio de fosfatase alcalina que foi maior nas mulheres (H= 68,72 UI/L; M=81,68 UI/L). Os níveis de ceruloplasmina (H=21,72mg/dL; M=27,78mg/dL) e de cobre sérico (H=71,38 ?g/dl; M=88,0 ug/dl) foram maiores em mulheres do que em homens (p<0,001). Os níveis de cuprúria basal foram 22,43ug/24h (média) e 21,40ug/24h (mediana); e, após a DPA, de 523,54 ug/24h (média) e 511,5 ug/24h (mediana). A cuprúria média basal masculina foi de 26,19. ug/24h, enquanto a feminina foi de 18,28. ug/24h (p=0,005). Com o presente estudo, ficou definida possível faixa de variação da cuprúria antes e depois da administração de dpenicilamina em pais de portadores com doença de Wilson; que os pais apresentaram cupremia e níveis de ceruloplasmina menores e cuprúria basal maior do que as mães; que as faixas de variação de normalidade para os parâmetros ceruloplasmina, cobre sérico e urinário deveriam ser diferenciadas de acordo com o sexo. / Wilson disease is a biliary copper excretion disturbance, of recessive autossomic heritage, due to ATP7B gene mutations. The copper not bound to apoceruloplasmin circulates in the organism bound to amino acids and accumulates mainly in the liver and brain being excreted by the kidneys. Urinary copper higher than 100ug/24h can be useful in the diagnosis, but only about 20% of Wilson disease patients have abnormal basal levels. In this case, d-penicillamine (DPA) administration can lead, in children, to levels higher than 1.600ug/24h. Twenty five fathers and twenty five mothers of wilson disease patients (mean 61 years for male, and 57 years for female) were assessed in order to obtain urinary copper levels of probable heterozygote adults. Fasting liver enzymes, copper and ceruloplasmin serum levels were obtained along with 24h urinary copper excretion. After, patients got DPA 1.0g by oral route, twice a day, while collecting urine for 24h urinary copper excretion dosage. These analyses were performed by elethrotermic atomic absortion spectrometry method. Liver enzyme levels were similar in men and women but those of alkaline phosphatase were higher in women (M= 68.72 UI/L; F=81.68 UI/L). Serum ceruloplasmin (F=21.72mg/dl; F=27.78mg/dl) and copper (M=71.38 ug/dl; F=88.0 ug/dl) levels were higher in women than in men (p<0.001). Urinary copper levels before DPA were, 43ug/24h (mean) and 21.40 ug/24h (median); and, after DPA, 523.54 ug/24h (mean) and 511.5 ug/24h (median). Basal urinary copper levels in men were 26.9 ug/24h, and in women were 18.67 ug/24h (p=0.005). With the results of this study, we defined a possible range for urinary copper before and after oral intake of DPA in parents of Wilson disease patients. Furthermore, fathers had lower levels of serum copper and of ceruloplasmin, and greater levels of baseline cupriuresis than mothers; and finally the normal range for serum copper and ceruloplasmin, and urinary copper should be differentiated according to the gender.