Gammaretroviral and lentiviral vectors for gene therapy: stability and inactivation mechanisms

Dissertation presented to obtain a Ph.D degree in Engineering and Technology Sciences, Gene Therapy at the Instituto de Tecnologia Quimica e Biológica, Universidade Nova de Lisboa

Plasma levels of tumor necrosis factor-alpha in patients with visceral leishmaniasis (Kala-Azar). Association with activity of the disease and clinical remisson following antimonial therapy

Evaluation of TNF-alpha in patients with Kala-azar has drawn increasing interest due to its regulatory role on the immune system, in addition to its cachetizing activity. The objective of this study was to examine the association between plasma levels of TNF-alpha, measured by immunore-activity (ELISA) and bioactivity (cytotoxicity assay with L-929 cells), and clinical manifestations of visceral leishmaniasis. Plasma samples from 19 patients with Kala-azar were obtained before, during and at the end of antimonial therapy. TNF-alpha determinations was done by using the cytotoxicity assay (all patients) and the enzyme-linked immunoassay (ELISA - 14 patients). A discrepancy between results obtained by ELISA and cytotoxicity assay was observed. Levels of circulating TNF-alpha, assessed by ELISA, were higher in patients than in healthy controls, and declined significantly with improvement in clinical and laboratory parameters. Plasma levels before treatment were 124.7 ± 93.3 pg/ml (mean ± SD) and were higher than at the end of therapy 13.9 ± 25.1 pg/ml (mean ± SD) (p = 0.001). In contrast, plasma levels of TNF-alpha evaluated by cytotoxicity assay did not follow a predicted course during follow-up. Lysis, in this case, might be not totally attributed to TNF-alpha. The discrepancy might be attributed to the presence of factor(s) known to influence the release and activity of TNF-alpha.

Identification of Toxoplasma gondii antigens involved in the IgM AND IgG indirect hemagglutination tests for the diagnosis of toxoplasmosis

Crude Toxoplasma gondii antigens represent raw material used to prepare reagents to be employed in different serologic tests for the diagnosis of toxoplasmosis, including the IgM and IgG indirect hemagglutination (IgG-HA and IgM-HA) tests. So far, the actual antigenic molecules of the parasite involved in the interaction with agglutinating anti-T. gondii antibodies in these tests are unknown. The absorption process of serum samples from toxoplasmosis patients with the IgG-HA reagent (G-toxo-HA) demonstrated that red cells from this reagent were coated with T. gondii antigens with Mr of 39, 35, 30, 27, 22 and 14 kDa. The immune-absorption process with the IgM-HA reagent (M-toxo-HA), in turn, provided antibody eluates which recognized antigenic bands of the parasite corresponding to Mr of 54, 35 and 30 kDa, implying that these antigens are coating red cells from this reagent. The identification of most relevant antigens for each type of HA reagent seems to be useful for the inspection of the raw antigenic material, as well as of reagent batches routinely produced. Moreover the present findings can be used to modify these reagents in order to improve the performance of HA tests for the diagnosis of toxoplasmosis

REACTIONAL STATES IN MULTIBACILLARY HANSEN DISEASE PATIENTS DURING MULTIDRUG THERAPY

It is well known that reactions are commonplace occurrences during the course of leprosy disease. Stigmatization may even be attributable to reactions which are also responsible for the worsening of neural lesions. A cohort of 162 newly-diagnosed baciloscopically positive patients from the Leprosy Care Outpatient Clinic of the Oswaldo Cruz Foundation (FIOCRUZ) was selected for this study. While 46% of the multibacillary (MB) patients submitted to the 24 fixed-dose multidrug therapy (MDT) regimen suffered reactions during treatment, it was found that all MBs were susceptible and that constant attention and care were required at all times. Fourteen per cent were classified as BB, 52% as BL, and 33% as LL. None of the variables under study, such as, sex, age, clinical form, length of illness, length of dermatological lesions, baciloscopic index (BI), or degree of disability proved to be associate with reaction among the patients studied. Reversal Reaction (RR) occurred in 45%, and Erythema Nodosum Leprosum (ENL) occurred in 55%. Among BB patients who developed reactions (15 patients), 93% presented RR; while among the LL patients who developed reactions (34 patients), 91% presented ENL. Likewise, ENL was very frequent among those with disseminate lesions, while RR was most often observed in patients with segmentary lesions. RR was also most likely to occur during the initial months of treatment. It was demonstrated that the recurrence rate of ENL was significantly higher than that of RR. Neither grade of disability nor BI was shown to be associated with RR and ENL reaction. However, the RR rate was significantly higher among patients showing BI < 3, while ENL predominated among those patients with BI > 3.

WIDESPREAD CUTANEOUS LESIONS DUE TO Sporothrix schenckii IN A PATIENT UNDER A LONG TERM STEROIDS THERAPY

A case of sporotrichosis in a woman presenting 63 cutaneous lesions distributed all over the tegument is related. The patient had both humoral (Immunoglobulins) and cellular (Lymphocytes subpopulations) immunity within normal limits, but was under treatment with steroid during a long time (Prednisone 10 mg daily for 2 years), due to a sciatic pain. In addition a review of the Brazilian literature on this type of lesions was carried out and commented.

TOXOCARIASIS: SEROLOGICAL DIAGNOSIS BY INDIRECT ANTIBODY COMPETITION ELISA

Toxocariasis is caused by infection of man by Toxocara canis and Toxocara cati larvae, the common roundworm of dogs and cats. Because larvae are difficult to detect in tissues, diagnosis is mostly based on serology. Non specific reactions are observed mainly due to cross-reactivity with Ascaris sp antigens. This investigation aimed at developing and evaluating an indirect antibody competition ELISA (IACE) employing a specific rabbit IgG anti-Toxocara canis excretory-secretory antigens as the competition antibody, in order to improve indirect ELISA specificity performed for toxocariasis diagnosis. For that, the rabbit IgG was previously absorbed by Ascaris suum adult antigens. Sensitivity and specificity of IACE were first evaluated in 28 serum samples of mice experimentally infected with T. canis embryonated eggs. Adopting cut-off value established in this population before infection, sensitivity and specificity were 100% after 20 days post-inoculation. For human population IACE was evaluated using sera from 440 patients with clinical signs of toxocariasis and the cut-off value was established with 60 serum samples from apparently healthy individuals. Using as reference test the indirect ELISA performed by Adolfo Lutz Institute, sensitivity was 60.2%, specificity was 98% and concordance was 77.3%. Repeatability of IACE was evaluated by the inter-reactions variation coefficient (2.4%).

Efficiency of indirect immunofluorescence assay as a confirmatory test for the diagnosis of human retrovirus infection (HIV-1 and HTLV-I/II) in different at risk populations

We compared the indirect immunofluorescence assay (IFA) with Western blot (Wb) as a confirmatory method to detect antibodies anti retrovirus (HIV-1 and HTLV-I/II). Positive and negative HIV-1 and HTLV-I/II serum samples from different risk populations were studied. Sensitivity, specificity, positive, negative predictive and kappa index values were assayed, to assess the IFA efficiency versus Wb. The following cell lines were used as a source of viral antigens: H9 ( HTLV-III b); MT-2 and MT-4 (persistently infected with HTLV-I) and MO-T (persistently infected with HTLV-II). Sensitivity and specificity rates for HIV-1 were 96.80% and 98.60% respectively, while predictive positive and negative values were 99.50% and 92.00% respectively. No differences were found in HIV IFA performance between the various populations studied. As for IFA HTLV system, the sensitivity and specificity values were 97.91% and 100% respectively with positive and negative predictive values of 100% and 97.92%. Moreover, the sensitivity of the IFA for HTLV-I/II proved to be higher when the samples were tested simultaneously against both antigens (HTLV-I-MT-2 and HTLV-II-MO-T). The overall IFA efficiency for HIV-1 and HTLV-I/II-MT-2 antibody detection probed to be very satisfactory with an excellent correlation with Wb (Kappa indexes 0.93 and 0.98 respectively). These results confirmed that the IFA is a sensitive and specific alternative method for the confirmatory diagnosis of HIV-1 and HTLV-I/II infection in populations at different levels of risk to acquire the infection and suggest that IFA could be included in the serologic diagnostic algorithm.

Chronic hepatitis C virus infections in Brazilian patients: association with genotypes, clinical parameters and response to long term alpha interferon therapy

The present study assessed the clinical significance of hepatitis C virus (HCV) genotypes and their influence on response to long term recombinant-interferon-alpha (r-IFN-a) therapy in Brazilian patients. One hundred and thirty samples from patients previously genotyped for the HCV and with histologically confirmed chronic hepatitis C (CH-C) were evaluated for clinical and epidemiological parameters (sex, age, time of HCV infection and transmission routes). No difference in disease activity, sex, age or mode and time of transmission were seen among patients infected with HCV types 1, 2 or 3. One hundred and thirteen of them were treated with 3 million units of r-IFN-a, 3 times a week for 12 months. Initial response (IR) was significantly better in patients with genotype 2 (100%) and 3 (46%) infections than in patients with genotype 1 (29%) (p < 0.005). Among subtypes, difference in IR was observed between 1b and 2 (p < 0.005), and between 1b and 3a (p < 0.05). Sustained response (SR) was observed in 12% for (sub)type 1a, 13% for 1b, 19% for 3a, and 40% for type 2; significant differences were found between 1b and 2 (p < 0.001), and between 1b and 3a (p < 0.05). Moreover, presence of cirrhosis was significantly associated with non response and response with relapse (p < 0.05). In conclusion, non-1 HCV genotype and lack of histological diagnosis of cirrhosis were the only baseline features associated with sustained response to treatment. These data indicate that HCV genotyping may have prognostic relevance in the responsiveness to r-IFN-a therapy in Brazilian patients with chronic HCV infection, as seen in other reports worldwide.

Nutritonal evaluation of pemphigus foliaceus patients on long term glucocorticoid therapy

Our objective was to compare food intake and nutritional status of Pemphigus Foliaceus patients (PG) on long term glucocorticoid therapy to a Control Group (CG). Fourteen PG female inpatients receiving prednisone (0.33 ± 0.22mg/kg) for at least 12 months and twelve CG subjects were submitted to nutritional evaluation, including anthropometry, urinary creatinine determination and serum biochemical measurements, besides 48-h-based food intake records. Groups were compared by Chi-square, Mann-Whitney and "t" tests. PG patients and CG were paired, respectively, in relation to age (24.7 ± 14.1 vs. 22.0 ± 12.0 years), body mass index (25.8 ± 6.4 vs. 24.0 ± 5.6kg/m2), daily protein intake (132.9 ± 49.8 vs. 95.2 ± 58.9g), and serum albumin (median; range) (3.8; 3.5-4.1 vs. 3.8; 3.6-5.0g/dl). However, PG patients had lower height-creatinine index (64.8 ± 17.6 vs. 90.1 ± 33.4%), and higher daily energy (3080 ± 1099 vs. 2187 ± 702kcal) and carbohydrate (376.8 ± 135.8 vs. 242.0 ± 80.7g) intakes. Despite high food, protein and energy consumption, PG patients on long term glucocorticoid therapy had lower body muscle mass than controls, while showing high body fat stores. These findings are possibly related to combined metabolic effects of long term corticotherapy and inflammatory disease plus corticosteroid-induced increased appetite.

Randomized, double-blind trial comparing indinavir alone, zidovudine alone and indinavir plus zidovudine in antiretroviral therapy-naive hiv-infected individuals with CD4 cell counts between 50 and 250/mm3

Treatment with indinavir has been shown to result in marked decreases in viral load and increases in CD4 cell counts in HIV-infected individuals. A randomized double-blind study to evaluate the efficacy of indinavir alone (800 mg q8h), zidovidine alone (200 mg q8h) or the combination was performed to evaluate progression to AIDS. 996 antiretroviral therapy-naive patients with CD4 cell counts of 50-250/mm3 were allocated to treatment. During the trial the protocol was amended to add lamivudine to the zidovudine-containing arms. The primary endpoint was time to development of an AIDS-defining illness or death. The study was terminated after a protocol-defined interim analysis demonstrated highly significant reductions in progression to a clinical event in the indinavir-containing arms, compared to the zidovudine arm (p<0.0001). Over a median follow-up of 52 weeks (up to 99 weeks), percent reductions in hazards for the indinavir plus zidovudine and indinavir groups compared to the zidovudine group were 70% and 61%, respectively. Significant reductions in HIV RNA and increases in CD4 cell counts were also seen in the indinavir-containing groups compared to the zidovudine group. Improvement in both CD4 cell count and HIV RNA were associated with reduced risk of disease progression. All three regimens were generally well tolerated.

Evaluation of penicillin therapy in patients with leptospirosis and acute renal failure

The effectiveness of specific antibiotic treatment in severe leptospirosis is still under debate. As part of a prospective study designed to evaluate renal function recovery after leptospirosis acute renal failure (ARF) (ARF was defined as Pcr > or = 1.5 mg/dL), the clinical evolutions of 16 treated patients (T) were compared to those of 18 untreated patients (nT). Treatment or non-treatment was the option of each patient's attending infectologist. The penicillin treatment was always with 6 million IU/day for 8 days. No difference was found between the two groups in terms of age, gender, number of days from onset of symptoms to hospital admission, or results of laboratory tests performed upon admission and during hospitalization, but proteinuria was higher in the treated group. There were no significant difference in the other parameters employed to evaluate patients' clinical evolution as: length of hospital stay, days of fever, days to normalization of renal function, days to total bilirubins normalized or reached 1/3 of maximum value and days to normalization of platelet counts. Dialytic treatment indication and mortality were similar between group T and nT. In conclusion, penicillin therapy did not provide better clinical outcome in patients with leptospirosis and ARF.