Interaction of alcohol and smoking in the pathogenesis of upper digestive tract cancers - possible chemoprevention with cysteine

Background: Alcohol consumption and smoking are the main causes of upper digestive tract cancers. These risk factors account for over 75% of all cases in developed countries. Epidemiological studies have shown that alcohol and tobacco interact in a multiplicative way to the cancer risk, but the pathogenetic mechanism behind this is poorly understood. Strong experimental and human genetic linkage data suggest that acetaldehyde is one of the major factors behind the carcinogenic effect. In the digestive tract, acetaldehyde is mainly formed by microbial metabolism of ethanol. Acetaldehyde is also a major constituent of tobacco smoke. Thus, acetaldehyde from both of these sources may have an interacting carcinogenic effect in the human upper digestive tract. Aims: The first aim of this thesis was to investigate acetaldehyde production and exposure in the human mouth resulting from alcohol ingestion and tobacco smoking in vivo. Secondly, specific L-cysteine products were prepared to examine their efficacy in the binding of salivary acetaldehyde in order to reduce the exposure of the upper digestive tract to acetaldehyde. Methods: Acetaldehyde levels in saliva were measured from human volunteers during alcohol metabolism, during tobacco smoking and during the combined use of alcohol and tobacco. The ability of L-cysteine to eliminate acetaldehyde during alcohol metabolism and tobacco smoking was also investigated with specifically developed tablets. Also the acetaldehyde production of Escherichia coli - an important member of the human microbiota - was measured in different conditions prevailing in the digestive tract. Results and conclusions: These studies established that smokers have significantly increased acetaldehyde exposure during ethanol consumption even when not actively smoking. Acetaldehyde exposure was dramatically further increased during active tobacco smoking. Thus, the elevated aerodigestive tract cancer risk observed in smokers and drinkers may be the result of the increased acetaldehyde exposure. Acetaldehyde produced in the oral cavity during ethanol challenge was significantly decreased by a buccal L-cysteine -releasing tablet. Also smoking-derived acetaldehyde could be totally removed by using a tablet containing L-cysteine. In conclusion, this thesis confirms the essential role of acetaldehyde in the pathogenesis of alcohol- and smoking-induced cancers. This thesis presents a novel experimental approach to decrease the local acetaldehyde exposure of the upper digestive tract with L-cysteine, with the eventual goal of reducting the prevalence of upper digestive tract cancers.

Affected sibling pairs with juvenile idiopathic arthritis : An immunogenetic study of the disease in multicase families

Genetic susceptibility to juvenile idiopathic arthritis (JIA) was studied in the genetically homogeneous Finnish population by collecting families with two or three patients affected by this disease from cases seen in the Rheumatism Foundation Hospital. The number of families ranged in different studies from 37 to 45 and the total number of patients with JIA, from among whom these cases were derived, was 2 000 to 2 300. Characteristics of the disease in affected siblings in Finland were compared with a population-based series and with a sibling series from the United States. A thorough clinical and ophthalmological examination was made of all affected patients belonging to sibpair series. Information on the occurrence of chronic rheumatic diseases in parents was collected by questionnaire and diagnoses were confirmed from hospital records. All patients, their parents and most of the healthy sibs were typed for human leukocyte antigen (HLA) alleles in loci A, C, B, DR and DQ. The HLA allele distribution of the cases was compared with corresponding data from Finnish bone marrow donors. The genetic component in JIA was found to be more significant than previously believed. A concordance rate of 25% for a disease with a population prevalence of 1 per 1000 implied a relative risk of 250 for a monozygotic (MZ) twin. An estimate for the sibling risk of an affected individual was about 15- to 20-fold. The disease was basically similar in familial and sporadic cases; the mean age at disease onset was however lower in familial cases, (4.8 years vs 7.4 years). Three sibpairs (3.4 expected) were concordant for the presence of asymptomatic uveitis. Uveitis would thus not appear to have any genetic component of its own, separate from the genetic basis of JIA. Four of the parents had JIA (0.2 cases expected), four had a type of rheumatoid factor-negative arthritis similar to that seen in juvenile patients but commencing in adulthood, and one had spondyloarthropathy (SPA). These findings provide additional support for the conception of a genetic predisposition to JIA and suggest the existence of a new disease entity, JIA of adult onset. Both the linkage analysis of the affected sibpairs and the association analysis of nuclear families provided overwhelming evidence of a major contribution of HLA to the genetic susceptibility to JIA. The association analysis in the Finnish population confirmed that the most significant associations prevailed for DRB1*0801, DQB1*0402, as expected from previous observations, and indicated the independent role of Cw*0401.

Part I: Enzyme replacement versus neurotrophic factor viral vector-mediated gene therapy of Parkinson’s disease, Part II: The effects of orally dosed tolcapone and entacapone on dopamine metabolism in the dorsal striatum and nucleus accumbens in rats

Part I: Parkinson’s disease is a slowly progressive neurodegenerative disorder in which particularly the dopaminergic neurons of the substantia nigra pars compacta degenerate and die. Current conventional treatment is based on restraining symptoms but it has no effect on the progression of the disease. Gene therapy research has focused on the possibility of restoring the lost brain function by at least two means: substitution of critical enzymes needed for the synthesis of dopamine and slowing down the progression of the disease by supporting the functions of the remaining nigral dopaminergic neurons by neurotrophic factors. The striatal levels of enzymes such as tyrosine hydroxylase, dopadecarboxylase and GTP-CH1 are decreased as the disease progresses. By replacing one or all of the enzymes, dopamine levels in the striatum may be restored to normal and behavioral impairments caused by the disease may be ameliorated especially in the later stages of the disease. The neurotrophic factors glial cell derived neurotrophic factor (GDNF) and neurturin have shown to protect and restore functions of dopaminergic cell somas and terminals as well as improve behavior in animal lesion models. This therapy may be best suited at the early stages of the disease when there are more dopaminergic neurons for neurotrophic factors to reach. Viral vector-mediated gene transfer provides a tool to deliver proteins with complex structures into specific brain locations and provides long-term protein over-expression. Part II: The aim of our study was to investigate the effects of two orally dosed COMT inhibitors entacapone (10 and 30 mg/kg) and tolcapone (10 and 30 mg/kg) with a subsequent administration of a peripheral dopadecarboxylase inhibitor carbidopa (30 mg/kg) and L- dopa (30 mg/kg) on dopamine and its metabolite levels in the dorsal striatum and nucleus accumbens of freely moving rats using dual-probe in vivo microdialysis. Earlier similarly designed studies have only been conducted in the dorsal striatum. We also confirmed the result of earlier ex vivo studies regarding the effects of intraperitoneally dosed tolcapone (30 mg/kg) and entacapone (30 mg/kg) on striatal and hepatic COMT activity. The results obtained from the dorsal striatum were generally in line with earlier studies, where tolcapone tended to increase dopamine and DOPAC levels and decrease HVA levels. Entacapone tended to keep striatal dopamine and HVA levels elevated longer than in controls and also tended to elevate the levels of DOPAC. Surprisingly in the nucleus accumbens, dopamine levels after either dose of entacapone or tolcapone were not elevated. Accumbal DOPAC levels, especially in the tolcapone 30 mg/kg group, were elevated nearly to the same extent as measured in the dorsal striatum. Entacapone 10 mg/kg elevated accumbal HVA levels more than the dose of 30 mg/kg and the effect was more pronounced in the nucleus accumbens than in the dorsal striatum. This suggests that entacapone 30 mg/kg has minor central effects. Also our ex vivo study results obtained from the dorsal striatum suggest that entacapone 30 mg/kg has minor and transient central effects, even though central HVA levels were not suppressed below those of the control group in either brain area in the microdialysis study. Both entacapone and tolcapone suppressed hepatic COMT activity more than striatal COMT activity. Tolcapone was more effective than entacapone in the dorsal striatum. The differences between dopamine and its metabolite levels in the dorsal striatum and nucleus accumbens may be due to different properties of the two brain areas.

Role of Areca Nut Induced TGF-beta and Epithelial-Mesenchymal Interaction in the Pathogenesis of Oral Submucous Fibrosis

Areca nut consumption has been implicated in the progression of Oral Submucous fibrosis (OSF); an inflammatory precancerous fibrotic condition. Our previous studies have demonstrated the activation of TGF-beta signaling in epithelial cells by areca nut components and also propose a role for epithelial expressed TGF-beta in the pathogenesis of OSF. Although the importance of epithelial cells in the manifestation of OSF has been proposed, the actual effectors are fibroblast cells. However, the role of areca nut and TGF-beta in the context of fibroblast response has not been elucidated. Therefore, to understand their role in the context of fibroblast response in OSF pathogenesis, human gingival fibroblasts (hGF) were treated with areca nut and/or TGF-beta followed by transcriptome profiling. The gene expression profile obtained was compared with the previously published transcriptome profiles of OSF tissues and areca nut treated epithelial cells. The analysis revealed regulation of 4666 and 1214 genes by areca nut and TGF-beta treatment respectively. The expression of 413 genes in hGF cells was potentiated by areca nut and TGF-beta together. Further, the differentially expressed genes of OSF tissues compared to normal tissues overlapped significantly with areca nut and TGF-beta induced genes in epithelial and hGF cells. Several positively enriched pathways were found to be common between OSF tissues and areca nut + TGF-beta treated hGF cells. In concordance, areca nut along with TGF-beta enhanced fibroblast activation as demonstrated by potentiation of alpha SMA, gamma SMA and collagen gel contraction by hGF cells. Furthermore, TGF-beta secreted by areca nut treated epithelial cells influenced fibroblast activation and other genes implicated in fibrosis. These data establish a role for areca nut influenced epithelial cells in OSF progression by activation of fibroblasts and emphasizes the importance of epithelial-mesenchymal interaction in OSF.

GFAP Isoforms in Adult Mouse Brain with a Focus on Neurogenic Astrocytes and Reactive Astrogliosis in Mouse Models of Alzheimer Disease

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Intranasal Delivery of Plasma and Platelet Growth Factors Using PRGF-Endoret System Enhances Neurogenesis in a Mouse Model of Alzheimer’s Disease

[EN] Neurodegeneration together with a reduction in neurogenesis are cardinal features of Alzheimer’s disease (AD) induced by a combination of toxic amyloid-β peptide (Aβ) and a loss of trophic factor support. Amelioration of these was assessed with diverse neurotrophins in experimental therapeutic approaches. The aim of this study was to investigate whether intranasal delivery of plasma rich in growth factors (PRGF-Endoret), an autologous pool of morphogens and proteins, could enhance hippocampal neurogenesis and reduce neurodegeneration in an amyloid precursor protein/presenilin-1 (APP/PS1) mouse model. Neurotrophic and neuroprotective actions were firstly evident in primary neuronal cultures, where cell proliferation and survival were augmented by Endoret treatment. Translation of these effects in vivo was assessed in wild type and APP/PS1 mice, where neurogenesis was evaluated using 5-bromodeoxyuridine (BdrU), doublecortin (DCX), and NeuN immunostaining 5 weeks after Endoret administration. The number of BrdU, DCX, and NeuN positive cell was increased after chronic treatment. The number of degenerating neurons, detected with fluoro Jade-B staining was reduced in Endoret-treated APP/PS1 mice at 5 week after intranasal administration. In conclusion, Endoret was able to activate neuronal progenitor cells, enhancing hippocampal neurogenesis, and to reduce Aβ-induced neurodegeneration in a mouse model of AD.

Control of bacterial disease in small scale fresh-water aquaculture: Project R0754. Project completion report December 1998 - May 1999

The work presented here represents an 18-month study to examine the relationship between environmental conditions, bacterial load in the water and bacteria levels in tissue macrophages of a range of clinically healthy freshwater fish species, farmed in a range of culture systems in Thailand and Vietnam. Preliminary assessment was made of the clinical significance of the macrophage bacterial load. The aim of this work was to improve production in fresh-water aquaculture through the control of clinical bacterial disease and subclinical infection, and to identify management practices most effective in promoting fish health. [PDF contains 37 pages]

A novel form of human disease with a protease-sensitive prion protein and heterozygosity methionine/valine at codon 129: Case report

Background: Sporadic Creutzfeldt-Jakob disease (sCJD) is a rare neurodegenerative disorder in humans included in the group of Transmissible Spongiform Encephalopathies or prion diseases. The vast majority of sCJD cases are molecularly classified according to the abnormal prion protein (PrPSc) conformations along with polymorphism of codon 129 of the PRNP gene. Recently, a novel human disease, termed "protease-sensitive prionopathy", has been described. This disease shows a distinct clinical and neuropathological phenotype and it is associated to an abnormal prion protein more sensitive to protease digestion. Case presentation: We report the case of a 75-year-old-man who developed a clinical course and presented pathologic lesions compatible with sporadic Creutzfeldt-Jakob disease, and biochemical findings reminiscent of "protease-sensitive prionopathy". Neuropathological examinations revealed spongiform change mainly affecting the cerebral cortex, putamen/globus pallidus and thalamus, accompanied by mild astrocytosis and microgliosis, with slight involvement of the cerebellum. Confluent vacuoles were absent. Diffuse synaptic PrP deposits in these regions were largely removed following proteinase treatment. PrP deposition, as revealed with 3F4 and 1E4 antibodies, was markedly sensitive to pre-treatment with proteinase K. Molecular analysis of PrPSc showed an abnormal prion protein more sensitive to proteinase K digestion, with a five-band pattern of 28, 24, 21, 19, and 16 kDa, and three aglycosylated isoforms of 19, 16 and 6 kDa. This PrPSc was estimated to be 80% susceptible to digestion while the pathogenic prion protein associated with classical forms of sporadic Creutzfeldt-Jakob disease were only 2% (type VV2) and 23% (type MM1) susceptible. No mutations in the PRNP gene were found and genotype for codon 129 was heterozygous methionine/valine. Conclusions: A novel form of human disease with abnormal prion protein sensitive to protease and MV at codon 129 was described. Although clinical signs were compatible with sporadic Creutzfeldt-Jakob disease, the molecular subtype with the abnormal prion protein isoforms showing enhanced protease sensitivity was reminiscent of the "protease-sensitive prionopathy". It remains to be established whether the differences found between the latter and this case are due to the polymorphism at codon 129. Different degrees of proteinase K susceptibility were easily determined with the chemical polymer detection system which could help to detect proteinase-susceptible pathologic prion protein in diseases other than the classical ones.

Avaliação da eficácia da terapia fotodinâmica como adjunto ao tratamento periodontal de pacientes com periodontite agressiva

A periodontite agressiva é um processo inflamatório de origem bacteriana mediado pelo sistema imunológico do hospedeiro e é provavelmente a forma mais grave de doença periodontal, apresentando destruição das estruturas tanto de proteção quanto de suporte dentário, num período relativamente rápido, normalmente levando a perda prematura dos elementos dentários e, em alguns casos, terapia antimicrobiana adjunta é necessária em adição à terapia mecânica. O objetivo do presente ensaio clínico randomizado controlado foi avaliar o efeito clínico da terapia fotodinâmica como adjunto ao tratamento periodontal não cirúrgico no tratamento da periodontite agressiva Os seguintes parâmetros clínicos foram avaliados: índice de placa visível, sangramento à sondagem, profundidade de bolsa à sondagem, nível de inserção clínica relativo, envolvimento de furca e mobilidade. Foram selecionados dez pacientes com periodontite agressiva, os quais foram examinados no dia zero e após três meses.O desenho do estudo consistiu em um modelo de boca dividida, onde um hemiarco foi tratado com raspagem e alisamento radicular e terapia fotodinâmica (laser diodo) e o outro apenas com raspagem e alisamento radicular. Três meses após o término do tratamento, os grupos terapêuticos apresentaram resultados semelhantes para todos os parâmetros clínicos avaliados: ambas as terapias tiveram sucesso, como redução de profundidade de bolsa, ganho de nível de inserção clínica relativo, redução de índice de placa visível, redução de sangramento à sondagem, diminuição de envolvimento de furca e diminuição de mobilidade, porém sem diferenças estatisticamente significantes entre elas. Dentro das limitações do presente estudo, os resultados sugerem que a terapia fotodinâmica adjuntamente ao tratamento periodontal não cirúrgico mecânico foi tão eficaz quanto o tratamento periodontal não cirúrgico mecânico sozinho.

Doença periodontal é fator de risco para parto prematuro e bebê de baixo peso?: revisão sistemática

O objetivo desta revisão sistemática foi analisar os estudos que relacionam doença periodontal e nascimento prematuro e baixo peso, verificando o efeito da doença periodontal materna no desfecho da gestação. Foi realizada pesquisa nas seguintes bases de dados: PubMed, Scielo e LILACS até Dezembro de 2007. A revisão sistemática foi conduzida de forma a identificar os estudos capazes de preencher os critérios de inclusão, apresentando aspectos clínicos, microbiológicos e radiográficos da doença periodontal, assim como os desfechos da gestação, ou seja, nascimento prematuro e baixo peso. Foram então selecionados 35 (trinta e cinco) artigos, sendo 9 (nove) coorte, 16 (dezesseis) casos controles, e 10 (dez) ensaios clínicos. A associação entre a doença periodontal materna e o nascimento de bebê prematuro com baixo peso foi encontrada em 26 (vinte e seis) estudos: 7 (sete) coorte, 11 (onze) casos controles e 8 (oito) ensaios clínicos. Não foi possível realizar uma meta-análise devido à grande heterogeneidade entre os estudos, particularmente no que se refere aos métodos de mensuração da doença periodontal. Um melhor controle em relação aos fatores de confusão também permitiria uma confiança maior nos resultados e conclusões apresentadas. Ainda não é possível ter conclusões adequadas do real efeito da doença periodontal sobre os desfechos da gestação devido a limitações nas metodologias dos presentes estudos. Portanto, a evidência da relação da doença periodontal com o nascimento de bebê prematuro e baixo peso é limitada. Ainda existe a necessidade de novos e bem desenhados estudos observacionais e de intervenção, que possam confirmar o que até agora é visto apenas como uma possível associação, explorando a validade dessas possíveis associações em diferentes populações e controlando adequadamente as variáveis de confusão.

Relação da doença periodontal com a anticoagulação oral e os benefícios do tratamento periodontal em pacientes anticoagulados

Anticoagulantes orais são amplamente indicados para prevenção de eventos tromboembólicos. No entanto, nem sempre os pacientes atingem a faixa terapêutica recomendada. Os objetivos desse estudo foram avaliar a associação entre periodontite e níveis de anticoagulação (fase 1) e o efeito do tratamento periodontal nos níveis de anticoagulação (fase 2) em pacientes que faziam uso do anticoagulante oral varfarina. O exame clínico incluiu índice CPO-D, índice de placa, sangramento à sondagem, profundidade de bolsa e nível de inserção clínica. Coeficiente normalizado internacional (INR), níveis de albumina, proteína C-reativa (PCR) e fibrinogênio foram avaliados no dia zero e até 180 dias após tratamento periodontal. Na fase 1 do estudo foram examinados 62 pacientes (42 mulheres e 20 homens, com idade média de 50,8 9,2 anos). Observamos uma correlação negativa entre extensão e severidade da doença periodontal e índice de placa com valores de INR. Não houve associação entre diagnóstico periodontal e níveis de anticoagulação. Dentre os pacientes fora do alvo terapêutico, 87% apresentavam diagnóstico de periodontite, enquanto no grupo na faixa terapêutica apenas 56%. Participaram da fase 2 do estudo 26 pacientes com periodontite severa (15 mulheres e 11 homens, com idade média de 51,3 9,2 anos). O tratamento periodontal resultou em melhora significativa de todos os parâmetros periodontais e dos níveis de anticoagulação 30, 60 90 e 180 dias após conclusão da terapia periodontal. Não houve alteração significativa na dose semanal da varfarina. Foi observada redução significativa entre níveis séricos de albumina dos dia 90 e 180 após a terapia periodontal, quando comparado aos valores do dia 0 (p < 0,05). De acordo com o alvo terapêutico estabelecido, observamos que no dia 0 doze pacientes (46,15%) estavam fora dessa faixa. Esse percentual foi reduzido significativamente após tratamento periodontal, sendo 26,1% e 29,2% nos dias 60 e 90, respectivamente. Embora tenha ocorrido melhora nos níveis de anticoagulação, não houve alteração significativa nos níveis de PCR e fibrinogênio. Sendo assim, pacientes com periodontite severa podem apresentar dificuldade para atingir a faixa terapêutica e o tratamento periodontal pode resultar em benefícios na busca da anticoagulação plena. Novos estudos são necessários para avaliar se formas menos severas de doença periodontal também podem interferir com a varfarina.

Efeitos da terapia periodontal na função endotelial e na expressão de marcadores inflamatórios sistêmicos

A periodontite é uma doença infecciosa, crônica e altamente prevalente causando uma resposta inflamatória. A infecção e a inflamação são consideradas a base etiológica para o desenvolvimento da aterosclerose. Recentes estudos indicam que a periodontite severa pode influenciar o aumento de marcadores inflamatórios e de disfunção endotelial associados com o aumento de risco da doença coronariana e o acidente vascular cerebral. Embora alguns estudos tenham sugerido esta associação, os reais efeitos do tratamento da doença periodontal sobre a rede complexa de marcadores envolvidos na aterosclerose são pouco conhecidos. O objetivo deste estudo foi avaliar o efeito da terapia periodontal nos biomarcadores inflamatórios (TNF-α, fibrinogênio, PCRus, INFγ, IL-1β, IL-6 e IL-10), perfil lipídico (CT, HDL, LDL, TG, oxLDL e anti-oxLDL) e função endotelial (IMT) das artérias carótidas. Um total de trinta e dois indivíduos saudáveis sistemicamente e afetados pela periodontite severa (16 mulheres, 16 homens; 51,87 anos de idade), incluindo 17 sujeitos para o grupo teste e 15 sujeitos para o grupo controle foram recrutados para o estudo. A ultrassonografia das artérias carótidas e os níveis séricos inflamatórios foram avaliados no início, 40 e 100 dias após o tratamento periodontal não-cirúrgico, comparando tempo e grupos. O tratamento periodontal resultou em significante redução dos parâmetros da doença periodontal. O grupo que recebeu tratamento mostrou decréscimos significativos de oxLDL (P<0.0001), anti-oxLDL (P<0,0001), TNF-α (P<0,0001), fibrinogênio (P=0,008), INFγ (P<0,0001), IL-1β (P<0,0001), IL-6 (P<0,0001), IMT (P=0,006) e significante aumento de IL-10 (P<0,0001) após 100 dias do tratamento periodontal comparando com grupo controle. Entretanto, os resultados não foram significativos para PCRu (P=0,109), colesterol total (P=0,438), HDL (P=0,119), LDL (P=0,425) e triglicerídeos (P=0,939). Com base nestes resultados, o tratamento da periodontite pode melhorar o perfil inflamatório e a função endotelial, sendo uma importante ferramenta adicional para a prevenção das doenças cardiovasculares.