Hipotiroidismo Associado a Anticorpos Anti-Receptor da Hormona TSH com Ação Bloqueadora Determinada In Vitro

Thyroid-stimulating hormone-receptor autoantibodies normally causes hyperthyroidism. However, they might have blocking activity causing hypothyroidism. A 11-year-old girl followed due to type 1 diabetes mellitus, celiac disease and euthyroid lymphocytic thyroiditis at diagnosis. Two years after the initial evaluation, thyroid-stimulating hormone was suppressed with normal free T4; nine months later, a biochemical evolution to hypothyroidism with thyroid-stimulating hormone-receptor autoantibodies elevation was seen; the patient remained always asymptomatic. Chinese hamster ovary cells were transfected with the recombinant human thyroid-stimulating hormone -receptor, and then exposed to the patient's serum; it was estimated a 'moderate' blocking activity of these thyroid-stimulating hormone-receptor autoantibodies, and concomitantly excluded stimulating action. In this case, the acknowledgment of the blocking activity of the serum thyroid-stimulating hormone-receptor autoantibodies, supported the hypothesis of a multifactorial aetiology of the hypothyroidism, which in the absence of the in vitro tests, we would consider only as a consequence of the destructive process associated to lymphocytic thyroiditis.

DNA damage induced by acrylamide: roe of genetic polymorphisms in DNA damage levels

RESUMO:Em 1994 a acrilamida (AA) foi classificada pela IARC como um provável cancerígeno para o homem. Para além da utilização de AA em numerosas aplicações industriais, a AA está também presente numa grande variedade de alimentos ricos em amido e processados a temperaturas elevadas. Esta exposição através da ingestão de produtos alimentares despoletou elevadas preocupações ao nível do risco para a saúde pública e poderá implicar um risco adicional para o aparecimento de cancro. A glicidamida (GA), o metabolito epóxido formado a partir da oxidação da AA provavelmente através do citocromo P450 2E1, é considerada por vários estudos, o principal responsável pela carcinogenicidade da AA. Actualmente existe uma escassez de resultados relativamente aos mecanismos de genotoxicidade da AA e GA em células de mamífero. Por este motivo, o objectivo deste estudo centra-se na avaliação das consequências genéticas da exposição à AA e GA, recorrendo-se para tal ao uso de células de mamífero como modelo. Tendo como base este objectivo avaliou-se a citotoxicidade da AA e GA, através do ensaio do MTT, e realizaram-se dois testes citogenéticos, o teste das aberrações cromossómicas (CAs) e o teste da troca de cromátides irmãs (SCEs), de modo a avaliar as lesões de DNA induzidas por estes compostos em células de hamster Chinês V79. Os resultados globalmente mostraram que a GA é mais citotóxica e clastogénica do que a AA. No âmbito deste trabalho, foi também efectuada a quantificação de aductos específicos de DNA, nomeadamente N7-(2-carbamoil-2-hidroxietil)guanina (N7-GA-Gua) e N3-(2-carbamoil-2-hidroxietil)adenina (N3-GA-Ade). Os resultados obtidos permitem afirmar que os níveis de N7-GA-Gua e a concentração de GA apresentam uma relação linear dose-resposta. Foi também identificada uma óptima correlação entre os níveis de N7-GA-Gua e a frequência de troca de cromátides irmãs. Adicionalmente, e de forma a compreender os mecanismos de toxicidade da AA, estudaram-se os mecanismos dependentes da modulação do glutationo reduzido (GSH), nomeadamente da butionina sulfoximina (BSO), um inibidor da síntese de GSH, do GSH-monoetil estér (GSH-EE), um composto permeável nas células e que é intra-celularmente hidrolisado a GSH e ainda do GSH adicionado exogenamente ao meio de cultura, em células V79. Os resultados obtidos reforçaram o papel da modulação do GSH nos efeitos de citotoxicidade e clastogenicidade da AA. Para além dos estudos efetuados com células V79, procedeu-se também à determinação da frequência de SCEs, à quantificação de aductos específicos de DNA, bem como ao ensaio do cometa alcalino em amostras de dadores saudáveis expostos à AA e GA. Tanto os resultados obtidos através do ensaio das SCE, como pela quantificação de aductos específicos de DNA, ambos efectuados em linfócitos estimulados, originaram resultados comparáveis aos obtidos anteriormente para as células V79, reforçando a ideia de que a GA é bastante mais genotóxica do que a AA. Por outro lado, os resultados obtidos pelo ensaio do cometa para exposição à AA e GA mostraram que apenas esta última aumenta o nível das lesões de DNA. Outro objectivo deste trabalho, foi a identificação de possíveis associações existentes entre as lesões de DNA, quantificadas através do ensaio das SCEs e do cometa, e biomarcadores de susceptibilidade, tendo em conta os polimorfismos genéticos individuais envolvidos na destoxificação e nas vias de reparação do DNA (BER, NER, HRR e NHEJ) em linfócitos expostos à GA. Tal permitiu identificar associações entre os níveis de lesão de DNA determinados através do ensaio das SCEs, e os polimorfismos genéticos estudados, apontando para uma possível associação entre o GSTP1 (Ile105Val) e GSTA2 (Glu210Ala) e a frequência de SCEs. Por outro lado, os resultados obtidos através do ensaio do cometa sugerem uma associação entre as lesões de DNA e polimorfismos da via BER (MUTYH Gln335His e XRCC1 Gln39Arg) e da via NER (XPC Ala499val e Lys939Gln), considerando os genes isoladamente ou combinados. Estes estudos contribuem para um melhor entendimento da genotoxicidade e carcinogenicidade da AA e GA em células de mamífero, bem como da variabilidade da susceptibilidade individual na destoxificação e reparação de lesões de DNA provocadas pela exposição a estes xenobióticos alimentares. ----------- ABSTRACT:Acrylamide (AA) has been classified as a probable human carcinogen by IARC. Besides being used in numerous industrial applications, AA is also present in a variety of starchy cooked foods. This AA exposure scenario raised concerns about risk in human health and suggests that the oral consumption of AA is an additional risk factor for cancer. A considerable number of findings strongly suggest that the reactive metabolite glycidamide (GA), an epoxide generated presumably by cytochrome P450 2E1, plays a central role in AA carcinogenesis. Until now there are a scarcity of results concerning the mechanisms of genotoxicity of AA and GA in mammalian cells. In view of that, the study described in this thesis aims to unveil the genetic consequences of AA and GA exposure using mammalian cells as a model system. With this aim we evaluated the cytotoxicity of AA and GA using the MTT assay and subsequently performed two cytogenetic end-points: chromosomal aberrations (CAs) and sister chromatid exchanges (SCEs), in order to evaluate DNA damage induced by these compounds in V79 Chinese hamster cell line. The results showed that GA was more cytotoxic and clastogenic than AA. Within the scope of this thesis the quantification of specific DNA adducts were also performed, namely N7-(2-carbamoyl-2-hydroxyethyl)guanine (N7-GA-Gua) and N3-(2-carbamoyl-2-hydroxyethyl)adenine (N3-GA-Ade). Interestingly, the GA concentration and the levels of N7-GA-Gua presented a linear dose-response relationship. Further, a very good correlation between the levels of N7-GA-Gua and the extent of SCEs were observed. In order to understand the mechanisms of AA-induced toxicity, the modulation of reduced glutathione (GSH)-dependent mechanisms were studied, namely the evaluation of the effect of buthionine sulfoximine (BSO), an effective inhibitor of GSH synthesis, of GSH-monoethyl ester (GSH-EE), a cell permeable compound that is intracellularly hydrolysed to GSH and also of GSH endogenously added to culture medium,z in V79 cell line. The overall results reinforced the role of GSH in the modulation of the cytotoxic and clastogenic effects induced by AA.Complementary to the studies performed in V79 cells, SCEs, specific DNA-adducts and alkaline comet assay in lymphocytes from healthy donors exposed to AA and GA were also evaluated. Both, the frequency of SCE and the quantification of specific GA DNA adducts, produced comparable results with those obtained in V79 cell line, reinforcing the idea that GA is far more genotoxic than AA. Further, the DNA damaging potential of AA and GA in whole blood leukocytes evaluated by the alkaline comet assay, showed that GA, but not AA, increases DNA damage. Additionally, this study aimed to identify associations between DNA damage and biomarkers of susceptibility, concerning individual genetic polymorphisms involved in detoxification and DNA repair pathways (BER, NER, HRR and NHEJ) on the GA-induced genotoxicity assessed by the SCE assay and by the alkaline comet assay. The extent of DNA damage determined by the levels of SCEs induced by GA seems to be modulated by GSTP1 (Ile105Val) and GSTA2 (Glu210Ala) genotypes. Moreover, the results obtained from the comet assay suggested associations between DNA damage and polymorphisms of BER (MUTYH Gln335His and XRCC1 Gln399Arg) and NER (XPC Ala499Val and Lys939Gln) genes, either alone or in combination. The overall results from this study contribute to a better understanding of the genotoxicity and carcinogenicity of AA and GA in mammalian cells, as well as the knowledge about the variability in individual susceptibility involved in detoxification and repair of DNA damage due to these dietary xenobiotics.

Caracterização de estirpes Mycobacterium Tuberculosis da família Lisboa: análise de regiões de diferença e single nucleotide polymorphisms

Dissertação apresentada para a obtenção do Grau de Mestre em Genética Molecular e Biomedicina, pela Universidade Nova de Lisboa, Faculdade de Ciências e Tecnologia

Development of an affinity pair “Tag-Receptor” for recombinant protein expression and purification

Dissertação apresentada para a obtenção do Grau de Mestre em Biotecnologia, pela Universidade Nova de Lisboa, Faculdade de Ciências e Tecnologia

Serum lipids of pemphigus foliaceus patients on long-term glucocorticoid therapy

Endemic pemphigus foliaceus, and long-term corticotherapy may affect serum lipid levels. The aim of this study was to compare serum lipids of pemphigus foliaceus patients on glucocorticoid therapy to a healthy control group. Fifteen patients receiving prednisone (0.33 ± 0.22mg/kg) for at least 12 months and 15 controls were submitted to 48-h food intake records, anthropometry, and biochemical measurements. Data were compared by chi2, Mann-Whitney and Student "t" tests. The groups were matched for gender, age, weight, body mass index, arm circumference and triceps skin fold. No differences were observed in relation to energy, fat, protein and carbohydrate daily intakes, total cholesterol, HDL, LDL, uric acid, and serum creatinine levels. Pemphigus foliaceus patients had higher triglyceride [159 (64-371) vs. 100 (45-133) mg/dl], VLDL [32 (13-74) vs. 20 (9-114) mg/dl] and ESR [44 (9-87) vs. 7 (1-30) mm/h] levels than controls, probably due to metabolic effects of inflammatory disease and corticotherapy.

Avaliação da expressão de interleucina 1 beta (IL-1β) e antagonista do receptor de interleucina 1 (IL-1Ra) em pacientes com hanseníase

A hanseníase é uma doença infectocontagiosa espectral que acompanha-se por uma série de eventos imunológicos desencadeados pela resposta do hospedeiro frente ao agente etiológico, o Mycobacterium leprae. Evidências sugerem que a indução e manutenção da resposta imune/inflamatória na hanseníase estão vinculadas a interações de múltiplas células e fatores solúveis, particularmente através da ação de citocinas. Nesse estudo, foram mensurados níveis de IL-1β e IL-1Ra de 37 casos novos de hanseníase acompanhados ao longo do tratamento e 30 controles sadios pelo teste ELISA. A coleta de sangue periférico foi realizada em quatro tempos para os casos de hanseníase (pré-tratamento com PQT, 2ª dose, 6ª dose e pós-PQT) e em único momento para os controles. Na comparação dos níveis das moléculas de casos no pré-PQT e controles, houve diferença estatisticamente significativa somente para IL-1β. Nossos resultados sugerem a participação dessa citocina no processo imune/inflamatório.

Novel affinity pairs "tag-receptor" for the purification of fusion proteins

Fundação para a Ciência e Tecnologia - SFRH/BD/48804/2008 and the project PTDC/BI/65383/2006 assigned to Prof. Cecíla Roque and also to Associate Laboratory REQUIMTE (Pest-C/EQB/LA0006/2011)

Characterization of mannose-binding lectin plasma levels and genetic polymorphisms in HIV-1-infected individuals

INTRODUCTION: The present study investigated the association between mannose-binding lectin (MBL) gene polymorphism and serum levels with infection by HIV-1. METHODS: Blood samples (5mL) were collected from 97 HIV-1-infected individuals resident in Belém, State of Pará, Brazil, who attended the Special Outpatient Unit for Infections and Parasitic Diseases (URE-DIPE). CD4+ T-lymphocyte count and plasma viral load were quantified. A 349bp fragment of exon 1 of the MBL was amplified via PCR, using genomic DNA extracted from controls and HIV-1-infected individuals, following established protocols. MBL plasma levels of the patients were quantified using an enzyme immunoassay kit. RESULTS: Two alleles were observed: MBL*O, with a frequency of 26.3% in HIV-1-infected individuals; and the wild allele MBL*A (73.7%). Similar frequencies were observed in the control group (p > 0.05). Genotype frequencies were distributed according to the Hardy-Weinberg equilibrium in both groups. Mean MBL plasma levels varied by genotype, with statistically significant differences between the AA and AO (p < 0.0001), and AA and OO (p < 0.001) genotypes, but not AO and OO (p = 0.17). Additionally, CD4+ T-lymphocytes and plasma viral load levels did not differ significantly by genotype (p > 0.05). CONCLUSIONS: The results of this study do not support the hypothesis that MBL gene polymorphism or low plasma MBL concentrations might have a direct influence on HIV-1 infection, although a broader study involving a large number of patients is needed.

Abnormal spontaneous interleukin 8 receptor expression: a brief report of two cases

Interleukin 8 (CXCL8) is an autocrine chemokine specific for the chemoattraction and activation of granulocytes, NKT cells and T lymphocytes. Patients with tuberculosis and latent Mycobacterium tuberculosis infection were assessed for the spontaneous expression of CXCR1 (CD128) and CXCR2 on lymphocytes and monocytes. Compared with ex vivo profiles, increased spontaneous CXCR2 expression and normal CXCR1 expression were found on lymphocytes in two out of 59 individuals. Monocytes showed normal ex vivo profiles for both receptors. After stimulation with purified protein derivative, the in vitro levels of CXCL8 were below the median levels of all patients with prior tuberculosis. Spontaneous CXCR2 modulation did not cause notable variation in the in vitro levels of CXCL8.

The effect of intravenous zoledronic acid on glucocorticoid-induced multiple vertebral fractures in juvenile systemic lupus erythematosus

Glucocorticoids are widely used in the treatment of lupus patients, and adverse effects, which include osteoporosis and associated fractures, are frequent. Treatment of osteoporosis of young patients should be effective and not harmful to bone growth and remodeling. Bisphosphonates are drugs that decrease the incidence of bone fractures, but their use in juvenile patients is still controversial because of their possible side effects on the growing skeleton. However, recently published studies showed that linear growth continued normally after treatment with these drugs, and there was no excessive suppression of bone remodeling or mineralization defects. Zoledronic acid is a new intravenous bisphosphonate that has been approved by the US FDA for use with hypercalcemia of malignancies and might be an effective treatment for postmenopausal osteoporosis. The authors report a case of a young girl with systemic lupus who developed multiple vertebral collapses due to glucocorticoid therapy, and zoledronic acid was used producing significant clinical and densitometric improvement.

Risk of multiple myeloma is associated with polymorphisms within telomerase genes and telomere length

Compelling biological and epidemiological evidences point to a key role of genetic variants of the TERT and TERC genes in cancer development. We analyzed the genetic variability of these two gene regions using samples of 2,267 multiple myeloma (MM) cases and 2,796 healthy controls. We found that a TERT variant, rs2242652, is associated with reduced MM susceptibility (OR?=?0.81; 95% CI: 0.72-0.92; p?=?0.001). In addition we measured the leukocyte telomere length (LTL) in a subgroup of 140 cases who were chemotherapy-free at the time of blood donation and 468 controls, and found that MM patients had longer telomeres compared to controls (OR?=?1.19; 95% CI: 0.63-2.24; ptrend ?=?0.01 comparing the quartile with the longest LTL versus the shortest LTL). Our data suggest the hypothesis of decreased disease risk by genetic variants that reduce the efficiency of the telomerase complex. This reduced efficiency leads to shorter telomere ends, which in turn may also be a marker of decreased MM risk.

Impact of TGF-ß1 -509C/T and 869T/C polymorphisms on glioma risk and patient prognosis

Transforming growth factor beta (TGF-ß) plays an important role in carcinogenesis. Two polymorphisms in the TGF-ß1 gene (-509C/T and 869T/C) were described to influence susceptibility to gastric and breast cancers. The 869T/C polymorphism was also associated with overall survival in breast cancer patients. In the present study, we investigated the relevance of these TGF-ß1 polymorphism in glioma risk and prognosis. A case-control study that included 114 glioma patients and 138 cancer-free controls was performed. Single nucleotide polymorphisms (SNPs) were evaluated by polymerase chain reaction followed by restriction fragment length polymorphism (PCR-RFLP). Univariate and multivariate logistic regression analyses were used to calculate odds ratio (OR) and 95 % confidence intervals (95 % CI). The influence of TGF-ß1 -509C/T and 869T/C polymorphisms on glioma patient survival was evaluated by a Cox regression model adjusted for patients' age and sex and represented in Kaplan-Meier curves. Our results demonstrated that TGF-ß1 gene polymorphisms -509C/T and 869T/C are not significantly associated with glioma risk. Survival analyses showed that the homozygous -509TT genotype associates with longer overall survival of glioblastoma (GBM) patients when compared with patients carrying CC + CT genotypes (OR, 2.41; 95 % CI, 1.06-5.50; p = 0.036). In addition, the homozygous 869CC genotype is associated with increased overall survival of GBM patients when compared with 869TT + TC genotypes (OR, 2.62; 95 % CI, 1.11-6.17; p = 0.027). In conclusion, this study suggests that TGF-ß1 -509C/T and 869T/C polymorphisms are not significantly associated with risk for developing gliomas but may be relevant prognostic biomarkers in GBM patients.

Risk factors, biochemical markers, and genetic polymorphisms in early coronary artery disease

OBJECTIVE: To assess the risk factors, lipid and apolipoprotein profile, hemostasis variables, and polymorphisms of the apolipoprotein AI-CIII gene in early coronary artery disease (CAD). METHODS: Case-control study with 112 patients in each group controlled by sex and age. After clinical evaluation and nutritional instruction, blood samples were collected for biochemical assays and genetic study. RESULTS: Familial history of early CAD (64 vs 39%), arterial hypertension (69 vs 36%), diabetes mellitus (25 vs 3%), and previous smoking (71 vs 46%) were more prevalent in the case group (p<0.001). Hypertension and diabetes were independent risk factors. Early CAD was characterized by higher serum levels of total cholesterol (235 ± 6 vs 209 ± 4 mg/dL), of LDL-c (154 ± 5 vs 135 ± 4 mg/dL), triglycerides (205 ± 12 vs 143 ± 9 mg/dL), and apolipoprotein B (129 ± 3 vs 105 ± 3 mg/dL), and lower serum levels of HDL-c (40 ± 1 vs 46 ± 1 mg/dL) and apolipoprotein AI (134 ± 2 vs 146 ± 2mg/dL) [p<0.01], in addition to an elevation in fibrinogen and D-dimer (p<0.02). The simultaneous presence of the rare alleles of the APO AI-CIII genes in early CAD are associated with hypertriglyceridemia (p=0.03). CONCLUSION: Of the classical risk factors, hypertension and diabetes mellitus were independently associated with early CAD. In addition to an unfavorable lipid profile, an increase in the thrombotic risk was identified in this population. An additive effect of the APO AI-CIII genes was observed in triglyceride levels.

Influence of angiotensin II receptor subtypes of the paraventricular nucleus on the physiological responses induced by angiotensin II injection into the medial septal area

OBJECTIVE: We determined the effects of losartan and PD 123319 (antagonists of the AT1 and AT2 angiotensin receptors, respectively), and [Sar¹, Ala8] ANG II (a relatively peptide antagonist of angiotensin receptors) injected into the paraventricular nucleus (PVN) on water and 3% NaCl intake, and the diuretic, natriuretic, and pressor effects induced by administration of angiotensin II (ANG II) into the medial septal area (MSA) of conscious rats. METHODS: Holtzman rats were used . Animals were anesthetized with tribromoethanol (20 mg) per 100 grams of body weight, ip. A stainless steel guide cannula was implanted into the MSA and PVN. All drugs were injected in 0.5-mul volumes for 10-15 seconds. Seven days after brain surgery, water and 3% NaCl intake, urine and sodium excretion, and arterial blood pressure were measured. RESULTS: Losartan (40 nmol) and [Sar¹, Ala8] ANG II (40 nmol) completely eliminated whereas PD 123319 (40 nmol) partially blocked the increase in water and sodium intake and the increase in arterial blood pressure induced by ANG II (10 nmol) injected into the MSA. The PVN administration of PD 123319 and [Sar¹, Ala8] ANG II blocked whereas losartan attenuated the diuresis and natriuresis induced by MSA administration of ANG II. CONCLUSION: MSA involvement with PVN on water and sodium homeostasis and arterial pressure modulation utilizing ANGII receptors is suggested.