EXPERIMENTAL INFECTION OF SWISS AND AKR/J MICE WITH Centrocestus formosanus (TREMATODA: HETEROPHYIDAE)

In order to better understand the biology of Centrocestus formosanus in a definitive host model, mice of Swiss and AKR/J strains were experimentally infected with 100 metacercariae of the parasite. Fourteen days post-infection, the rodents were killed and adult trematodes were recovered from the small intestine. The percentage of parasite recovery from AKR/J mice (11.4%) was significantly higher than that from Swiss mice (5.3%). Moreover, trematodes recovered from the AKR/J strain were more developed and had greater fecundity. Peculiarities concerning the mice’s immune system could explain the difference in susceptibility and in worm development seen in the present study. The data obtained confirm that mice are susceptible to infection with C. formosanus and indicate that the AKR/J strain provides a more favorable environment for parasite development.

EFFECT OF Bifidobacterium animalis ON MICE INFECTED WITH Strongyloides venezuelensis

The administration of viable Bifidobacterium animaliswas tested to induce resistance against Strongyloides venezuelensis infection in mice. Effects on parasite burden, worm length, egg output, and intestinal mucosal histology were evaluated. The oral administration of B. animalis, strain 04450B, starting 14 days before the inoculation of nematode larvae significantly decreased the worm burden and egg output. In probiotic treated animals, the percent reduction of adult worms in the intestine was of 33% and the reduction of egg production was of 21%, compared with those of the control group. The duodenum villous height and villous/crypt ratio were significantly higher in probiotic-treated mice, indicating that this group could be experiencing less intestinal damage. The present findings revealed that the administration of B. animalis for the amelioration of host response to nematode infections is biologically plausible and could have some potential for impacting public health. Meanwhile, further study is needed to delineate the nature and identity of the factor(s) involved in these beneficial effects.

HISTORICAL SERIES OF PATIENTS WITH VISCERAL LEISHMANIASIS TREATED WITH MEGLUMINE ANTIMONIATE IN A HOSPITAL FOR TROPICAL DISEASES, MACEIÓ-AL, BRAZIL

Introduction: Visceral leishmaniasis is an endemic protozoan found in Brazil. It is characterized by fever, pallor, hepatosplenomegaly, lymphadenopathy, and progressive weakness in the patient. It may lead to death if untreated. The drug of choice for treatment is meglumine antimoniate (Glucantime®). The aim of this study was to evaluate patients with visceral leishmaniasis according to criteria used for diagnosis, possible reactions to Glucantime® and blood pressure measured before and after treatment. Methods: 89 patients admitted to the Teaching Hospital Dr. Hélvio Auto (HEHA) in Maceió-AL, in the period from May 2006 to December 2009 were evaluated. Data were collected on age, sex, origin, method of diagnosis, adverse effects of drugs, duration of hospitalization, duration of treatment and dosage up to the onset of adverse effects. Results: There was a predominance of child male patients, aged between one and five years old, from the interior of the State of Alagoas. Parasitological diagnosis was made by bone marrow aspirate; three (3.37%) patients died, 12 (13.48%) had adverse reactions and treatment was changed to amphotericin B, and 74 (83.14%) were cured. Changes that led to replacing Glucantime® were persistent fever, jaundice, rash, bleeding and cyanosis. Conclusion: During the study, 89 patients hospitalized for VL were analyzed: 74 were healed, 12 were replaced by amphotericin B treatment and three died. Most of them were under five years old, male and came from the interior. The dosage and duration of treatment with Glucantime® were consistent with that advocated by the Ministry of Health. Persistence of fever, jaundice, rash, cyanosis and bleeding were the reactions that led the physician to modify treatment. No change was observed in blood pressure before and after treatment. This study demonstrated the work of a hospital, a reference in the treatment of leishmaniasis, which has many patients demanding its services in this area. It demonstrates that this disease is still important today, and needs to be addressed properly to prevent injury and death due to the disease.

ELEVATED TRANS-MAMMARY TRANSMISSION OF Toxocara canis LARVAE IN BALB/c MICE

Toxocariasis is a widespread zoonosis and is considered an important worldwide public health problem. The aim of this study was to investigate the frequency of trans-mammary Toxocara canis infection in newborn BALB/c mice nursed by females experimentally infected with 1,200 eggs after delivery. After 50 days of age, the presence of larvae in different organs of the offspring was investigated. Trans-mammary infection was confirmed in 73.9% of the mice that had been nursed by infected females. These data show a high trans-mammary transmission of T. canis and confirm the significance of this transmission route in paratenic hosts.

Epidemiology of Asthma and Allergic Diseases in Portuguese Speaking Regions

The application of the same epidemiological methods in different countries allows important comparisons between different races and cultures. During the last decade, two large multi-centres epidemiological studies, the Portuguese Study of Allergic Diseases in Childhood (PAC study) and the International Study of Asthma and Allergies in Childhood (ISAAC study), were implemented in Portuguese speaking regions. The main objectives were to assess and compare allergic diseases prevalence. The authors stress out the significant differences observed in schoolchildren from the three continents, with different genetic and environmental background. It was found an increase trend in the prevalence of all allergic diseases, mainly rhinitis, in last decade. Rhinitis has been identified as an independent risk factor for asthma in Caucasian population.

EFFICACY OF NITAZOXANIDE AGAINST Toxocara canis: LARVAL RECOVERY AND HUMORAL IMMUNE RESPONSE IN EXPERIMENTALLY INFECTED MICE

SUMMARY The efficacy of nitazoxanide (NTZ) against toxocariasis was investigated in an experimental murine model and results were compared to those obtained using mebendazole. Sixty male BALB/c mice, aged six to eight weeks-old, were divided into groups of 10 each; fifty were orally infected with 300 larvaed eggs of T. canisand grouped as follows, G I: infected untreated mice; G II: infected mice treated with MBZ (15 mg/kg/day) 10 days postinfection (dpi); G III: infected mice treated with NTZ (20 mg/kg/day) 10 dpi; G IV: infected mice treated with MBZ 60 dpi; G V: infected mice treated with NTZ 60 dpi; GVI: control group comprising uninfected mice. Mice were bled via retro-orbital plexus on four occasions between 30 and 120 dpi. Sera were processed using the ELISA technique to detect IgG anti- Toxocaraantibodies. At 120 dpi, mice were sacrificed for larval recovery in the CNS, liver, lungs, kidneys, eyes and carcass. Results showed similar levels of anti- ToxocaraIgG antibodies among mice infected but not submitted to treatment and groups treated with MBZ or NTZ, 10 and 60 dpi. Larval recovery showed similar values in groups treated with NTZ and MBZ 10 dpi. MBZ showed better efficacy 60 dpi, with a 72.6% reduction in the parasite load compared with NTZ, which showed only 46.5% reduction. We conclude that administration of these anthelmintics did not modify the humoral response in experimental infection by T. canis. No parasitological cure was observed with either drug; however, a greater reduction in parasite load was achieved following treatment with MBZ.

THE RELEVANCE OF NUTRITIONAL STATUS AND HISTOPATHOLOGICAL FINDINGS ON THE INFECTIOUS PROCESS OF BALB/C MICE INOCULATED WITH Lacazia loboi

SUMMARY The aim of this study was to evaluate the effects of the protein-calorie malnutrition in BALB/c isogenic mice infected with Lacazia loboi, employing nutritional and histopathological parameters. Four groups were composed: G1: inoculated with restricted diet, G2: not inoculated with restricted diet, G3: inoculated with regular diet, G4: not inoculated with regular diet. Once malnutrition had been imposed, the animals were inoculated intradermally in the footpad and after four months, were sacrificed for the excision of the footpad, liver and spleen. The infection did not exert great influence on the body weight of the mice. The weight of the liver and spleen showed reduction in the undernourished groups when compared to the nourished groups. The macroscopic lesions, viability index and total number of fungi found in the footpads of the infected mice were increased in G3 when compared to G1. Regarding the histopathological analysis of the footpad, a global cellularity increase in the composition of the granuloma was observed in G3 when compared to G1, with large numbers of macrophages and multinucleated giant cells, discrete numbers of lymphocytes were present in G3 and an increase was observed in G1. The results suggest that there is considerable interaction between Jorge Lobo's disease and nutrition.

PATHOGENICITY OF Blastocystis sp. TO THE GASTROINTESTINAL TRACT OF MICE: RELATIONSHIP BETWEEN INOCULUM SIZE AND PERIOD OF INFECTION

The pathogenic potential of Blastocystis sp. in experimental models requires further investigation. In this work, the pathogenicity of this parasite in the gastrointestinal tract of male Swiss mice was evaluated according to the inoculum size and period of infection. Animals were infected intragastrically, with 100, 500, 1,000, 5,000 and 10,000 Blastocystis sp. vacuolar forms obtained from a mixture of eight human isolates cultured axenically in Jones' medium. After seven, 14, 21, 28 and 60 days of infection, the animals were sacrificed and fragments of the small intestine (duodenum), large intestine, and cecum were subjected to histopathological analysis. Blastocystis sp. triggered an inflammatory response in the different tissues analyzed, with a predominance of mononuclear cells. The parasite was found in the muscular layer of the cecum, showing its invasive character. Larger inocula triggered inflammatory processes earlier (seven days) than smaller ones (from 21 days). We conclude that, in the proposed model, the pathogenicity of Blastocystis sp. isolates that were studied is related to inoculum size and period of infection.

CD19 LYMPHOCYTE PROLIFERATION INDUCED BY Bifidobacterium animalis subsp. lactis IN C57BL/6 MICE EXPERIMENTALLY INFECTED WITH Toxoplasma gondii

Toxoplasmosis is frequently acquired through the oral route by the ingestion of cysts or oocysts of Toxoplasma gondii. Once ingested, the parasites penetrate the intestinal epithelial cells and rapidly disseminate to all organs in the host. During T. gondii infection, the intestinal microbiota plays an important role in stimulating a protective immune response against the parasite. In this sense the use of probiotics is worthy of note since they are live microorganisms that have beneficial effects on the host through stimulation of the immune response that can be important in the control of T. gondii proliferation and dissemination in the host. In the present study, the action of the probiotic Bifidobacterium animalis subsp. lactis was investigated in C57BL/6 mice infected with oocysts of ME49 strain of T. gondii. The probiotic had an immunomodulatory action, inducing CD19 lymphocyte proliferation and consequently increasing anti-T. gondii antibody level.Bifidobacterium animalis subsp. lactisprovided protection in supplemented mice, compared to the control group. In addition, supplemented animals had milder inflammatory process in the small intestine, indicating that the probiotic protects the intestinal mucosa during infection with T. gondii. It was concluded that the probioticB. animalis subsp. lactis induces humoral immune response capable of providing protection against T. gondii infection.

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.

The effect of phenolated "vaccines" against experimental T. cruzi infection in mice

"Vaccines" prepared from parasites of an avirulent cultivated Y strain of T. cruzi, suspended in phenolated 1/10.000 saline solution, with aluminum stearate, containing alive parasites, gave high degree of protection to mice against a posterior infection with virulent blood forms of the same parasites and strain. The degree of protection with 1/1000 and 1/10.000 phenol "vaccines", with no alive parasites, was very poor specially in the first group. The immunity seems to be related to the number of alive trypanosomes in the "vaccines".

Thoracic Manifestations of Connective Tissue Diseases

Connective tissue diseases (CTDs) comprise several immunologic systemic disorders, each of which associated with a particular set of clinical manifestations and autoimmune profile. CTDs may cause numerous thoracic abnormalities, which vary in frequency and pattern according to the underlying disorder. The CTDs that most commonly involve the respiratory system are progressive systemic sclerosis, systemic lupus erythematosus, rheumatoid arthritis, Sjögren syndrome, polymyositis, dermatomyositis, and mixed connective tissue disease. Pulmonary abnormalities in this group of patients may result from CTD-related lung disease or treatment complications, namely drug toxicity and opportunistic infections. The most important thoracic manifestations of CTDs are interstitial lung disease and pulmonary arterial hypertension, with nonspecific interstitial pneumonia being the most common pattern of interstitial lung disease. High-resolution computed tomography is a valuable tool in the initial evaluation and follow-up of patients with CTDs. As such, general knowledge of the most common high-resolution computed tomographic features of CTD-related lung disease allows the radiologist to contribute to better patient management.

Splenectomy and experimental infection of mice by a virulent strain of trypanosoma cruzi

Splenectomy seems to increase susceptibility of mice to a jurther infection with a virulent strain of Trypanosoma cruzi. Parasitemia increases with splenectomy and the sooner the infection follows the operation, the greater the parasitemia. The mortality rate seems to have not been influenced by splenectomy.

Comparison of different techniques for mouse transgenesis

RESUMO: O uso de ratinhos transgénicos em neurociências aumentou consideravelmente nos últimos anos devido ao crescente interesse em compreender o cérebro e a necessidade de solucionar situações clínicas do foro neurológico e psiquiátrico. Para esse efeito, diferentes métodos de produção de animais transgénicos têm sido testados. O objectivo desta tese foi comparar métodos de integração aleatória de um transgene no genoma de ratinhos em termos de eficiência, estabilidade da integração do transgene, número de animais e de horas de trabalho necessárias para cada método. Assim, foi comparado o método mais utilizado - microinjecção pronuclear (PNMI) - com duas outras técnicas cujo desempenho foi considerado promissor – a transferência génica através dos testículos por electroporação e transfecção por lentivírus in vivo. As três técnicas foram realizadas usando um gene repórter sob o controlo de um promotor constitutivo, e depois reproduzidas usando um gene de interesse de modo a permitir obtenção de um animal capaz de ser usado em experimentação laboratorial. O transgene de interesse utilizado codifica uma proteína de fusão correspondendo a uma variante da rodopsina (channelrhodopsin) fundida à proteína enhanced yellow fluorescente protein ((EYFP) resultando num produto designado ChR2-EYFP. Este animal transgénico apresentaria expressão deste canal iónico apenas em células dopaminergicas, o que, com manipulação optogenética, tornaria possivel a activação especifica deste grupo de neurónios e, simultaneamente, a observação do impacto desta manipulação no comportamento num animal em livre movimento. Estas ferramentas são importantes na investigação básica em neurociências pois ajudam a esclarecer o papel de grupos específicos de neurónios e compreender doenças como a doença de Parkinson ou a esquizofrenia onde a função de certos tipos de neurónios de encontra alterada. Quando comparados os três métodos realizados verifica-se que usando um gene repórter PMNI resulta em 31,3% de, a de animais transgénicos obtidos, a electroporação de testículos em 0% e a injecção de lentivírus em 0%. Quando usado o gene de interesse, os resultados obtidos são, respectivamente, 18,8%, 63,9% e 0%.--------------ABSTRACT: The use of transgenic mice is increasing in all fields of research, particularly in neuroscience, due to the widespread need of animal models to solve neurological and psychiatric medical conditions. Different methodologies have been tested in the last decades in order to produce such transgenic animals. The ultimate goal of this thesis is to compare different methods of random integration of a transgene in the genome of mice in terms of efficiency, stability of the transgene integration, number of animals required and the labour intensity of each technique. We compared the most used method – pronuclear microinjection (PNMI) – with two other promising techniques – Testis Mediated Gene Transfer (TMGT) by electroporation and in vivo lentiviral transfection. The three techniques were performed using a reporter gene – green fluorescent protein (GFP), whose transcription was driven by the constitutive cytomegalovirus (CMV) promoter. These three techniques were later reproduced using the tyrosine hydroxylase promoter (TH) and the neuronal manipulator, channelrhodopsin-2 fused to the enhanced yellow fluorescent reporter protein (ChR2-EYFP). The transgenic animal we sough to produce would express the light driven channel only in dopaminergic cells, making possible to specifically activate this group of neurons, while simultaneously observe the behaviour in a freely moving animal. This is a very important tool in basic neuroscience research since it helps to clarify the role of specific groups of neurons, map circuits in the brain, and consequently understand neurological diseases such as Parkinson’s disease or schizophrenia, where the function of certain types of neurons is affected. When comparing the three methods, it was verified that using a reporter gene PNMI resulted in 31.3% of transgenic mice obtained, testis electroporation in 0% and lentiviral injection in 0%. When using the gene of interest, the results obtained were, respectively, 18.8%, 63.9% and 0%.