Evaluation of the ELISA-F29 test as an early marker of therapeutic efficacy in adults with chronic Chagas disease

This work compared the time at which negative seroconversion was detected by conventional serology (CS) and by the ELISA-F29 test on a cohort of chronic chagasic patients treated with nifurtimox or benznidazole. A retrospective study was performed using preserved serum from 66 asymptomatic chagasic adults under clinical supervision, and bi-annual serological examinations over a mean follow-up of 23 years. Twenty nine patients received trypanocide treatment and 37 remained untreated. The ELISA-F29 test used a recombinant antigen which was obtained by expressing the Trypanosoma cruzi flagellar calcium-binding protein gene in Escherichia coli. Among the untreated patients, 36 maintained CS titers. One patient showed a doubtful serology in some check-ups. ELISA-F29 showed constant reactivity in 35 out of 37 patients and was negative for the patient with fluctuating CS. The treated patients were divided into three groups according to the CS titers: in 13 they became negative; in 12 they decreased and in four they remained unchanged. ELISA-F29 was negative for the first two groups. The time at which negativization was detected was significantly lower for the ELISA-F29 test than for CS, 14.5 ± 5.7 and 22 ± 4.9 years respectively. Negative seroconversion was observed in treated patients only. The results obtained confirm that the ELISA-F29 test is useful as an early indicator of negative seroconversion in treated chronic patients.

EVALUATION OF THE THERAPEUTIC EFFICACY OF LEVAMISOLE HYDROCHLORIDE ON THIRD-STAGE LARVAE OF Lagochilascaris minor IN EXPERIMENTALLY INFECTED MICE

Lagochilascariosis, a disease caused by Lagochilascaris minor, affects the neck, sinuses, tonsils, lungs, the sacral region, dental alveoli, eyeballs and the central nervous system of humans. A cycle of autoinfection may occur in human host tissues characterized by the presence of eggs, larvae and adult worms. This peculiarity of the cycle hinders therapy, since there are no drugs that exhibit ovicidal, larvicidal and vermicidal activity. Given these facts, we studied the action of levamisole hydrochloride on third-stage larvae in the migration phase (G1) and on encysted larvae (G3) of L. minor. To this end, 87 inbred mice of the C57BL/6 strain were divided into test groups comprising 67 animals (G1-37; G3-30) and a control group (G2-10; G4-10) with 20 animals. Each animal was inoculated orally with 2,000 infective eggs of the parasite. The animals of the test groups were treated individually with a single oral dose of levamisole hydrochloride at a concentration of 0.075 mg. The drug was administered either 30 minutes prior to the parasite inoculation (G1 animals) or 120 days after the inoculation (G3 animals). The mice in the control groups were not treated with the drug. After the time required for the migration and the encysting of L. minor larvae, all the animals were euthanized and their tissues examined. The data were analyzed using the Student's unpaired t-test and the Levene test. The groups showed no statistically significant difference. Levamisole hydrochloride was ineffective on third-stage larvae of L. minor. These findings explain the massive expulsion of live adult worms, as well as the use of long treatment schemes, owing to the persistence of larvae and eggs in human parasitic lesions.

A Prospective Assessment of Renal Transplantation Versus Haemodialysis: Which Therapeutic Modality Is Good Value for Society?

Background: Economic evaluations help health authorities facing budget constraints. This study compares the health-related quality of life (HRQOL) and costs in patient subgroups on haemodialysis (HD) and renal transplantation (KT). Methods: In a prospective study with follow-up of 1-3 years, we performed a costutility analysis of KT vs. HD, adopting a lifetime horizon. A societal perspective was taken. Costs for organ procurement, KT eligibility, transplant surgery and follow-up of living donors were included. Key clinical events were recorded. HRQOL was assessed using the EuroQol instrument. Results: The HRQOL remained stable on HD patients. After KT, mean utility score improved at 3 months while mean EQ-VAS scores showed a sustained improvement. Mean annual cost for HD was 32,567.57€. Mean annual costs for KT in the year-1 and in subsequent years were, 60,210.09€ and 12,956.77€ respectively. Cost for initial hospitalization averaged 18,740.74€. HLA-mismatches increased costs by 75% for initial hospitalization (p < 0.001) and 41% in the year-1 (p < 0.05), and duplicate the risk of readmission in the year-1 (p < 0.05). The incremental costutility ratio was 5,534.46€/QALY, increasing 35% when costs for organ procurement were added. KT costs were 41,541.63€ more but provided additional 7.51 QALY. Conclusions: The KT is cost-effective compared with HD. Public funding should reflect the value created by the intervention and adapt to the organ demand.

Long-Term Lamivudine Treatment of Children with Chronic Hepatitis B: Durability of Therapeutic Responses and Safety

Lamivudine has been demonstrated safe and efficacious in the short term in a large cohort of children with chronic hepatitis B (CHB), but optimal duration of treatment has not been elucidated and limited data on the safety of long-term lamivudine administration have been reported. In addition, the durability of favourable therapeutic outcomes after lamivudine therapy in children has not been well characterized. The aim of this study was to examine the safety of lamivudine and the durability of clinical responses in a group of children who received up to 3 years of treatment for CHB. One hundred and fifty-one children from centres in nine countries who had previously received lamivudine in a large prospective trial were enrolled. During the first year, children had been randomized to either lamivudine or placebo treatment. Subsequently, in a separate extension study, those who remained hepatitis B e antigen (HBeAg) positive were given lamivudine for up to 2 years and those who were HBeAg negative were observed for additional 2 years. Results of these studies have been previously reported. In this study, these children were followed for 2 additional years. Data gathered from medical record review included weight, height, signs and symptoms of hepatitis, alanine aminotransferase (ALT) levels, serologic markers, hepatitis B virus (HBV) DNA levels and serious adverse events (SAEs). Other pharmacological treatments for CHB were allowed according to the practices of individual investigators and were documented. Subjects were divided into two groups for analysis, those who had achieved virological response (VR), defined as HBeAg negative and undetectable HBV DNA by the bDNA assay by the end of the extension study at 3 years, and those who had not. In those who had achieved VR by the end of the extension study, long-term durability of HBeAg seroconversion was 82% and >90% in those who had received lamivudine for 52 weeks and at least 2 years respectively. This compares to 75% for those who had achieved seroconversion after placebo. In those who had not achieved VR by the end of the extension study, an additional 11% did so by the end of the study; they had all received lamivudine in the previous trial, and none had received further treatment during the study. Eight children lost hepatitis B surface antigen during the study and all had received lamivudine at some point during the previous trials. Evaluation of safety data revealed no SAEs related to lamivudine. There was no effect of treatment on weight or height z scores. Clinically benign ALT flares (>10 times normal) were seen in 2% of children. Favourable outcomes from lamivudine treatment of CHB in children are maintained for at least several years after completion of treatment. Up to 3 years of lamivudine treatment is safe in children.

Therapeutic trial in experimental tegumentary leishmaniasis caused by Leishmania (Leishmania) amazonensis. A comparative study between mefloquine and aminosidine

One hundred and eighty-two male inbred C57/BL/6 mice were infected with 3 x 106 Leishmania (Leishmania) amazonensis promastigotes of the MHOM/BR/PH8 strain by means of a subcutaneous injection in the right ear. The animals were separated in three groups: 1) oral mefloquine hydrochloride treatment (16mg/kg/day/10 days), 2) intramuscular aminosidine (Paromomycin®) treatment (20mg/kg/20 days) and 3) control. Twenty six mice of each treated group were sacrificed, one at the end of treatment (nine weeks after inoculation), and one six weeks later (fifteen weeks after inoculation). Control Group animals were sacrificed at weeks six, nine and fifteen after inoculation. There was no significant difference between Group 1 (mefloquine) and Group 3 (control) subjects. Group 2 animals (aminosidine) presented the smallest differences of all, both at the end of the treatment and six weeks later. The histopato-logical parameters have shown the following findings: a) there was no significant difference between the mefloquine treated group and the control group; the group treated with aminosidine showed fewer of vacuolated macrophages than the control group, at week 9 (end of treatment). b) both at the end of treatment and six weeks later, evaluation of tissue necrosis and tissue fibrosis revealed no differences between the treated groups. It was found that six weeks after the end of treatment, mice in the control group presented significantly more severe degrees of fibrosis than mice in the other groups. It can be concluded that mefloquine showed limited therapeutic effect in this experimental model, whereas aminosidine had a significant effect. Nevertheless, neither of them resulted in cure of the lesions.

Successful therapeutic response of resistant cases of mucocutaneous leishmaniasis to a very low dose of antimony

Two mucocutaneous leishmaniasis cases resistant to therapy are reported here. After the failure of initial therapies (antimony, amphotericin B and/or pentamidine) patients received a low-dose schedule: one ampoule of meglumine antimoniate (405mg of pentavalent antimony [Sb v]) by intramuscular injection, three times a week until complete healing of the lesions. One patient was cured with a total of 30 ampoules in 10 weeks and the other received 36 ampoules in 12 weeks. Both remain clinically cured after one year of follow-up.

Trauma and envenoming caused by stingrays and other fish in a fishing community in Pontal do Paranapanema, state of São Paulo, Brazil: epidemiology, clinical aspects, and therapeutic and preventive measures

INTRODUCTION: Accidents caused by fish are common in inland fishing communities in Brazil, being work-related injuries in the majority of cases. These populations have no information on the mechanisms of trauma or envenoming. METHODS: Through a questionnaire administered to fishermen, we obtained clinical and epidemiological data on accidents in Rosana, Pontal do Paranapanema, State of São Paulo, Brazil. These data were analyzed and converted into an easily understood prevention and treatment program for the colony. RESULTS: Thirty-nine fishermen replied to the survey. All of the patients had been hurt by fish. Of those mentioned, the yellow catfish (Pimelodus maculatus) was the main fish species associated with injuries, but others also caused trauma to the fishermen. Six fishermen had been envenomed by stingrays. Pain and ulcers were the main symptoms and were described as intolerable. Approximately half of those injured were treated using traditional folk remedies. CONCLUSIONS: The fishermen suffered multiple accidents with catfish, which are venomous and cause intense pain, as well as trauma due to other fish, such as surubins, traíras, freshwater croakers, and piranhas. Approximately 16% of those interviewed presented with envenomation from stingrays. Our data and previous experience in the area led to the creation of a pamphlet with clear language that can effectively help fishermen in the region, an area in need of health services and disease prevention. This initiative also applies to the whole La Plata River basin, which has similar fauna.

Evaluation of the therapeutic response of hepatitis C in coinfected patients (HIV/HCV): a study of cases from a hospital for chronic liver diseases in the Eastern Brazilian Amazon

INTRODUCTION: The aim of this study was to evaluate the therapeutic response of hepatitis C in patients coinfected with human immunodeficiency virus (HIV-1). METHODS: A retrospective study of 20 patients coinfected with HIV-1/HCV who were treated in the outpatient liver clinic at the Sacred House of Mercy Foundation Hospital of Pará (Fundação Santa Casa de Misericórdia do Pará - FSCMPA) from April 2004 to June 2009. Patients were treated with 180µg PEG interferon-α2a in combination with ribavirin (1,000 to 1,250mg/day) for 48 weeks. The end point was the sustained virological response (SVR) rate (HCV RNA negative 24 weeks after completing treatment). RESULTS: The mean age of the patients was 40±9.5 years, of which 89% (n=17) were male, and the HCV genotypes were genotype 1 (55%, n=11/20), genotype 2 (10%, n=2/20) and genotype 3 (35%, n=7/20). The mean CD4+ lymphocyte count was 507.8, and the liver fibrosis stages were (METAVIR) F1 (25%), F2 (55%), F3 (10%) and F4 (10%). The early virological response (EVR) was 60%, the end-of-treatment virological response (EOTVR) was 45% and the SVR was 45%. CONCLUSIONS: The median HCV viral load was high, and in 85% of cases in which highly active antiretroviral therapy (HAART) was used, none of the patients with F3-F4 fibrosis responded to treatment. Of the twenty patients treated, 45% achieved SVR and 45% achieved EOTVR. Studies that include cases from a wider region are needed to better evaluate these findings.

Phage display as a tool for generation of bio-therapeutic agents for breast cancer

Notch is a conserved signalling pathway, which plays a crucial role in a multiple cellular processes such as stem cell self-renewal, cell division, proliferation and apoptosis. In mammalian, four Notch receptors and five ligands are described, where interaction is achieved through their extracellular domains, leading to a transcription activation of different target genes. Increased expression of Notch ligands has been detected in several types of cancer, including breast cancer suggesting that these proteins represent possible therapeutic targets. The goal of this work was to generate quality protein targets and, by phage display technology, select function-blocking antibodies specific for Notch ligands. Phage display is a powerful technique that allows the generation of highly specific antibodies to be used for therapeutics, and it has also proved to be a reliable approach in identifying and validating new cancer-related targets. Also, we aimed at solving the tri-dimensional structure of the Notch ligands alone and in complex with selected antibodies. In this work, the initial phase focused on the optimization of the expression and purification of a human Delta-like 1 ligand mutant construct (hDLL1-DE3), by refolding from E. coli inclusion bodies. To confirm the biological activity of the produced recombinant protein cellular functional studies were performed, revealing that treatment with hDLL1-DE3 protein led to a modulation of Notch target genes. In a second stage of this study, Antibody fragments (Fabs) specific for hDLL1-DE3 were generated by phage display, using the produced protein as target, in which one good Fab candidate was selected to determine the best expression conditions. In parallel, multiple crystallization conditions were tested with hDLL1-DE3, but so far none led to positive results.

Narrative change in Gloria Films: Comparing various processes of therapeutic innovation

Aims: The present study focuses on the analysis of novelty emergence in classic Gloria Films with Rogers, Perls, and Ellis to understand how the same client formulated her own problem and if and how change occurred in those three sessions. Method: The Innovative Moments Coding System was applied to track innovative moments (IMs) and their themes. Results: The session with Rogers showed more diversity in disclosed problems and themes of IMs, as well as a higher proportion of reflection IMs. The session with Perls demonstrated a high proportion of protest IMs. The session with Ellis showed less innovation than other sessions. The changes found were based mostly on reflection and protest IMs in three sessions. Conclusion: Narrative innovations occurred in the three single sessions. The type of dominant innovation is consistent with the therapeutic model and the IMs model. The exploration of the IMs’ themes allowed a more precise identification of Gloria's new narrative positions and their development throughout those sessions.

Dissolution enhancement of active pharmaceutical ingredients by therapeutic deep eutectic systems

A therapeutic deep eutectic system (THEDES) is here defined as a deep eutectic solvent (DES) having an active pharmaceutical ingredient (API) as one of the components. In this work, THEDESs are proposed as enhanced transporters and delivery vehicles for bioactive molecules. THEDESs based on choline chloride (ChCl) or menthol conjugated with three different APIs, namely acetylsalicylic acid (AA), benzoic acid (BA) and phenylacetic acid (PA), were synthesized and characterized for thermal behaviour, structural features, dissolution rate and antibacterial activity. Differential scanning calorimetry and polarized optical microscopy showed that ChCl:PA (1:1), ChCl:AA (1:1), menthol:AA (3:1), menthol:BA (3:1), menthol:PA (2:1) and menthol:PA (3:1) were liquid at room temperature. Dissolution studies in PBS led to increased dissolution rates for the APIs when in the form of THEDES, compared to the API alone. The increase in dissolution rate was particularly noticeable for menthol-based THEDES. Antibacterial activity was assessed using both Gram-positive and Gram-negative model organisms. The results show that all the THEDESs retain the antibacterial activity of the API. Overall, our results highlight the great potential of THEDES as dissolution enhancers in the development of novel and more effective drug delivery systems.

Protein-based nanodevices for therapeutic applications

Tese de Doutoramento em Biologia Molecular e Ambiental (área de especialização em Biologia Celular e Saúde).

Biosurfactants produced by marine microorganisms with therapeutic applications

Marine microorganisms possess unique metabolic and physiological features and are an important source of new biomolecules, such as biosurfactants. Some of these surface-active compounds synthesized by marine microorganisms exhibit antimicrobial, anti-adhesive and anti-biofilm activity against a broad spectrum of human pathogens (including multi-drug resistant pathogens), and could be used instead of the existing drugs to treat infections caused by them. In other cases, these biosurfactants show anti-cancer activity, which could be envisaged as an alternative to conventional therapies. However, marine biosurfactants have not been widely explored, mainly due to the difficulties associated with the isolation and growth of their producing microorganisms. Culture-independent techniques (metagenomics) constitute a promising approach to study the genetic resources of otherwise inaccessible marine microorganisms without the requirement of culturing them, and can contribute to discover novel biosurfactants with significant biological activities. This paper reviews the most relevant biosurfactants produced by marine microorganisms with potential therapeutic applications and discusses future perspectives and opportunities to discover novel molecules from marine environments.

A possible contribution to improving the therapeutic potentials of Babor's Typology of Alcohol Dependent Patients

ABSTRACT Objective The objective of this study was to replicate Babor's Typology and to explore clinical features related to personality traits that may underlie this classification, in order to improve its therapeutic possibilities. Methods Observational prospective study on a group of 273 male alcoholics. After a replication of Babor's variables, Cluster Analysis, Chi-Square – applied on clinical variables related to a Lappda Tipology – and Kappa tests were performed. Results The study identified two distinct clusters that held similar features to those described for the Type A/Type B classification. Besides presenting a lower socio-economic situation, Cluster 2 patients were associated with higher vulnerability and severe clinical features and also differed from Cluster 1 in their response to treatment. These replicated clusters retained connections and also differences in relation to the variables derived from the Lappda Typology. Conclusion Considering that each of the two replicated clusters seem to be associated to different personality traits – according to their correlations to the affective, cognitive and behavioral dimensions brought forward by the Lappda Typology – it is acceptable that this study may contribute to the development of more comprehensive and effective therapeutic strategies specifically tailored to target more specific personality traits of these subgroups of alcoholic patients.

Searching for therapeutic strategies in a mouse modelo of Machado-Joseph disease: targeting proteostases

Tese de Doutoramento em Ciências da Saúde