Homogneous time resolved fluorescence: a new technique for detection and quantifications of infliximab in autoimmune patients

RESUMO:As terapias biológicas revolucionaram o tratamento das doenças autoimunes nos últimos anos. Tipicamente têm como alvos mediadores importantes no mecanismo das doenças. Os antagonistas do fator de necrose tumoral-α (TNF-α) são um grupo de agentes biológicos muito prescrito, pois estão indicados no tratamento de doenças imuno-mediadas comuns, tais como artrite reumatoide, artrite idiopática juvenil, artrite psoriática, espondilite anquilosante, doença de Crohn e colite ulcerosa. Com o uso frequente de inibidores do TNF-α, tem-se tornado evidente que estes agentes têm um potencial imunogénico importante, que pode comprometer o prognóstico a longo prazo dos doentes cronicamente tratados. A produção de anticorpos anti-fármaco parece causar falência terapêutica secundária em muitos doentes. Um dos efeitos dos anticorpos anti-fármaco é o aumento da eliminação do fármaco. A eliminação do fármaco, por sua vez, varia entre indivíduos, refletindo diferentes perfis farmacocinéticos. A determinação dos níveis séricos mínimos do agente anti-TNF-α é assim muito informativa e pode auxiliar nas decisões terapêuticas. Contudo, os testes imunológicos para determinar as concentrações séricas do fármaco não estão facilmente disponíveis na prática clínica. De forma a investigar uma nova técnica potencialmente fidedigna e prática para a deteção e quantificação dos agentes biológicos anti-TNF-α, foi testada a técnica por HTRF (homogeneous time-resolved fluorescence resonance energy transfer) para a determinação de concentrações séricas de infliximab. Apesar de apresentar algumas limitações relacionadas com as condições de leitura da fluorescência, esta técnica provou obter resultados próximos das concentrações obtidas por ELISA (enzyme-linked immunosorbent assay) bridging. Adicionalmente, tem a vantagem de ser de execução muito mais fácil e rápida. Deste modo, a técnica por HTRF poderá ser otimizada e tornar-se uma valiosa ferramenta laboratorial para orientar as decisões terapêuticas em doentes autoimunes com falência da terapêutica anti-TNF-α.--------- ABSTRACT: Biologic therapies revolutionized the treatment of autoimmune diseases in the last years. Typically, they target important disease mediators. Tumor necrosis factor-alpha (TNF-α) antagonists constitute a very prescribed group of biologic agents as they are indicated for the treatment of common immune-mediated diseases, such as rheumatoid arthritis, juvenile idiopathic arthritis, psoriatic arthritis, ankylosing spondylitis, Crohn’s disease and ulcerative colitis. With the increasing use of TNF-α inhibitors it has been noticed that they have an important immunogenic potential that can compromise long-term outcomes in chronically treated patients. The production of anti-drug antibodies seems to cause secondary therapeutic failure in many patients. One of the effects of anti-drug antibodies is the enhancement of drug clearance. Drug clearance, in turn, varies among individuals, reflecting different pharmacokinetic profiles. Determination of serum anti-TNF-α drug trough levels is though very informative and could support treatment decisions. However, immunologic assays to determine drug serum concentrations are not readily available in clinical practice. In order to investigate a potentially reliable and practical new technique for detection and quantification of anti-TNF-α biologic agents, homogeneous time-resolved fluorescence resonance energy transfer (HTRF) technique was tested for determination of serum infliximab concentrations. Although presenting some limitations related with fluorescence reading conditions, this technique proved to give results close to the concentrations obtained by the widely used bridging enzyme-linked immunosorbent assay (ELISA). In addition, it has the advantage of being much easier and faster to perform. Thus, HTRF technique can be optimized and become a valuable laboratorial tool to guide treatment decisions in autoimmune patients with anti-TNF-α therapy failure.

Symptomless Plasmodium vivax parasitemias and malaria eradication in Santa Catarina State, Brazil

Thirty-nine symptomless carriers of P. vivax parasites in the blood gave blood films at monthly intervals for four to six months during the non- transmission season. It was found that parasitaemias can continue for many months. Thirteen of those studied relapsed with symptoms and were treated with chloroquine at a dosage of 600 mg for adults with proportionate doses for children. Of these nine relapsed silently while under observation: a symptomless relapse rate of approximately 70 per cent. One case had symptoms attributable to malaria close to the time of the original survey (the day before). Of the remaining 38 asymptomatic parasite carriers four showed microgametocytes in a density that suggested a potentially high infectivity and six showed microgametocytes in a density suggesting a potentially low to moderate infectivity for mosquito vectors. There was thus a proportion of one smptomatic case of malaria to 10 potentially infective symptomless parasite carriers. Because they feel no need to seek treatment, such persons may form an important reservoir of infection when vectors cannot be fully controlled by spraying. Some possible methods of dealing with such situations are discussed.

Label-free detection of biomolecules with Ta2O5-based field effect devices

Dissertação para obtenção do Grau de Doutor em Nanotecnologias e Nanociências

Effects of lethal and non-lethal malaria on the mononuclear phagocyte system

The effects ofone non-lethal species ofmalarialparasite, Plasmodium yoelii, and one lethal species, P. berghei, on the mononuclear phagocyte system (MPS) of BALB/c mice were studied. P. yoelii caused a greater and more sustained expansion and activation of the MPS, and the two major populations of spleen phagocytic cells-red pulp and marginal zone macrophages - exhibited a greater increase in numbers in this infection. During the course of P. berghei mataria, the spleen was progressively occupied by haematopoietic tissue and, at the terminal stage of infection, an extensive depletion of lymphocytes and macrophages was apparent. The possibility was suggested that the outcome of mataria may be inftuenced by the particular way the parasite interacts with the MPS.

Sensitivity of Gomez-Nuñez boxes for the detection of household infestation with Panstrongylus megistus

Four methods for detecting household infestations with Panstrongylus megistus were compared: 1) manual collection; 2) collection after pyrethrum application; 3) search viable eggs; and 4) Gomez-Nuñez boxes. Manual collection was the most sensitive method (23% infested), followed by pyrethrum (21%), Gomez-Nuñez boxes (15%) and viable eggs (12%). About 10% of infested houses were positive exclusively on the Gomez-Nuñez box test. More over, 6 out of the 7 houses positive exclusively on the Gomez-Nuñez method were located in a recently sprayed area, where P. megistus density was low. Inspection of Gomez-Nuñez boxes at 12 weekspost-application was twice as effective as inspection at 6 weekspost-application. Triatomine feces was the most common evidencefor thepresence of P. megistus found within Gomez-Nuñez boxes. Gomez-Nuñez boxes area a useful adjunct to manual collection in detecting domestic infestations with P. megistus, especially in areas where bug densities are low. However, the utility of Gomez-Nuhez boxes must be weighed against the time and labor they require.

The role of spleen macrophages in malaria: an ultrastructural study

An electronmicroscopy study of the spleen from mice infected with Plasmodium berghei was carried out to investigate the types ofcells in volved in the removal of parasites from the blood, and the mechanisms by which this occurs. Macrophages, particularly from the red pulp and the marginal zone of the spleen, constituted the most important population of phagocytic cells in the spleen. At the height ofparasitaemia, macrophages in the periphery of the white pulp, especially in the mantle zone of secondary follicles, were also found to participate in phagocytosis, although to a limited extent. Our fingings suggest that phagocytosis of free parasites or parasitized erythrocytes in the spleen is an important mechanism of clearance of parasites from the circulation. Parasites removed from the erythrocytes when these cells cross the interendothelial slits are further phagocytosed by neighbouring macrophages. Evidence is presented suggesting that spleen macrophages may act against the parasite through a process of cytotoxicity.

Frequency of human leukocyte antigen (hla) in patients with malaria and in the general population of Humaitá county, Amazonas state, Brazil

In August 1983,85 inhabitants of the municipality of Humaitá, Amazonas State, Brazil were studied to determine the prevalence of antigens to HLA-A, -B, -C and DR. Thirty-eight were sick with malaria due to Plasmodium falciparum. All subjects were examined for splenomegaly, blood parasitaemia and antibodies to malaria. They constituted three groups: 1) 25 subjects native to the Amazon region who had never had malaria; 2) 38 Amazonian subjects who had malaria in the past or currently had an infection; 3) 22 patients with malaria who had acquired the infection in the Amazon Region but came from other regions of Brazil. Blood was taken from each person, the lymphocytes were separated and typed by the test of microlymphocytotoxicity. There was a high frequency of antigens that could not be identified in the groups studied which suggests the existence of a homozygote or phenotype not identified in the population. There was a high frequency of the phenotype Ag(W24) (44.7%) in group 2 when compared with group 1 (32%) or group 3 (9%). Also the individuals in group 2 showed an elevated frequency of antigen DR(4)80%) when compared with group 1 (36.6%) or group 3 (16.6%). These observations suggest the possibility of a genetic susceptibility to malaria among Amazonian residents and indicate a necessity for more extensive studies of the frequency of HLA antigens among inhabitants of this endemic malarial zone.

Infrequency of asymptomatic malaria in an endemic area in Amazonas, Brazil

A malaria survey was conducted in an area of high transmission (Costa Marques, Rondonia, Brazil) to determine the prevalence of asymptomatic parasitemia and its clinical significance. Most of the people surveyed were immigrants who had lived in the endemic area < 5 years. The people had easy access to free diagnostic and treatment services at the Malaria Clinic in the town of Costa Marques. The prevalence of plasmodial parasitemia in 344 people was 22%. There were 36 individuals with asymptomatic infections among the 77 parasitemic patients. During the two days following the initial examination, 19 ofthe 36 individuals: with asymptomatic infections developed malaria. Among the 17 patients who remained asymptomatic for > 2 days, 4 had only gametocytes, 1 had taken inadequate anti-malarial treatment, 3 were under treatment and 2 moved. Six asymptomatic patients denied the use of anti-malarial drugs and they developed malaria 3-6 days after the initial parasitological diagnosis. The final patient remained asymptomatic during the 7 day observation period. He had a history of > 40 malaria attacks and denied the use of antimalarial treatment. With the exception of the latter all of the other asymptomatic patients, were either in the incubation period or had been treated It is concluded that asymptomatic malaria is rare in the Costa Marques area and that it is necessary to treat all individuals with plasmodial parasitemia.

Malaria in humait a county, state of Amazonas, Brazil. XIX - evaluation of clindamycin for the treatment of patients with Plasmodium falciparum infection

A total of 207 patients with malaria caused by Plasmodium falciparum were submitted to 5 different treatment schedules with clindamycin from 1981 to 1984: A - 89 patients were treated intravenously and orally, or intramuscularly and orally with 20 mg/kg/day divided into two daily applications for 5 to 7 days; B-40 patients were treated orally with 20 mg/kg/day divided into two daily doses for 5 to 7 days; C-27 patients were treated with 20 mg/kg/day intravenously or orally divided into two daily applications for 3 days; D-16 patients were treated orally and/or intravenously with a single daily dose of 20 to 40 mg/kg/day for 5 to 7 days; E-35 patients were treated orally with 5 mg/kg/day divided into two doses for 5 days. Patients were examined daily during treatment and reexamined on the 7th, 24th, 21st, 28th and 35th day both clinically and parasitologically (blood test). Eighty three (40.1%) had moderate or severe malaria, and 97 (46.8%) had shown resistance to chloroquine or to the combination ofsulfadoxin and pyrimethamine. The proportion of cured patients was higher than 95% among patients submitted to schedules A and B. Side effects were only occasional and of low intensity. Three deaths occurred (1.4%), two of them involving patients whose signs and symptoms were already very severe when treatment was started. Thus, clindamycin proved to be very useful in the treatment of patients with malaria caused by Plasmodium falciparum and we recommend schedule A for moderate and severe cases and Bfor initial cases.

Time series morphological analysis applied to biomedical signals events detection

Dissertation submitted in the fufillment of the requirements for the Degree of Master in Biomedical Engineering

Two competitive enzyme immunoassays for the detection of IgG class antibodies to hepatitis a antigen

Two competitive enzyme immunoassays (EIA) techniques were developed: in the first (COMP-1), test sera were added together with HAV antigen on anti-HAV IgG-coated wells followed by an anti-HA VHRP conjugate; in the second (COMP-2), test sera and anti-HA VHRP conjugate competed for HAV epitopes previously adsorbed to anti-HA V IgG-coated wells. Both procedures used tetramethylbenzidine (TMB) as a substrate. Both competitive tests were shown to be reproducible and suitable for routine diagnosis and research purposes.

Reduction of spleen size in a child with Hyperreactive Malarious Splenomegaly (HMS) treated outside the Brazilian endemic area of malaria with only one course of quinine

We report the clinical picture, treatment and evolution of a child with hyperreactive malarious splenomegaly treated outside the endemic area of malaria. The patient presented gross splenomegaly, proceeded from an area where malaria is endemic, showed increased immunoglobulins levels, high antimalarial antibody titres and hepatic sinusoidal lymphocytosis. The child did not return to an area where malaria is endemic and showed a favorable response to only one course of quinine. The response of this patient to limited antimalarial therapy suggests the importance of reinfection with malaria in the development and maintenance of this syndrome.