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The objective of this study was to evaluate the disposition kinetics of enrofloxacin ( ENR) in the plasma and its distribution in the muscle tissue of Nile tilapia ( Oreochromis niloticus) after a single oral dose of 10 mg/kg body weight via medicated feed. The fish were kept at a temperature between 28 and 30 °C. The collection period was between 30 min and 120 h after administration of the drug. The samples were analyzed by high-performance liquid chromatography with a fluorescence detector ( HPLC- FLD). The ENR was slowly absorbed and eliminated from the plasma ( Cmax = 1.24 ± 0.37 μg/mL; Tmax = 8 h; T1/2 Ke = 19.36 h). ENR was efficiently distributed in the muscle tissue and reached maximum values (2.17 ± 0.74 μg/g) after 8 h. Its metabolite, ciprofloxacin ( CIP), was detected and quantified in the plasma (0.004 ± 0.005 μg/mL) and muscle (0.01 ± 0.011 μg/g) for up to 48 h. After oral administration, the mean concentration of ENR in the plasma was well above the minimum inhibitory concentrations ( MIC50) for most bacteria already isolated from fish except for Streptococcus spp. This way the dose used in this study allowed for concentrations in the blood to treat the diseases of tilapia. [ABSTRACT FROM AUTHOR]

Mucosal surfaces have a fundamental participation in many aspects of the human immunodeficiency virus (HIV) infection pathogenesis. In Brazilian HIV-1 infected subjects, loss of weight and appetite are among the most debilitating symptoms. In this review we describe a defined mucosal immunogen that has profound but transient effects on HIV viral load, and we suggest that gut associated lymphoid tissue under constant immunostimulation is likely to provide a major contribution to the total levels of HIV. We also show that hypermetabolism appears to play a role in the wasting process in Brazilian patients coinfected with HIV and tuberculosis.

Abstract Background Bacille Calmette-Guerin (BCG) is currently the only available vaccine against tuberculosis (TB) and comprises a heterogeneous family of sub-strains with genotypic and phenotypic differences. The World Health Organization (WHO) affirms that the characterization of BCG sub-strains, both on genomic and proteomic levels, is crucial for a better comprehension of the vaccine. In addition, these studies can contribute in the development of a more efficient vaccine against TB. Here, we combine two-dimensional electrophoresis (2DE) and mass spectrometry to analyse the proteomic profile of culture filtrate proteins (CFPs) from M. bovis BCG Moreau, the Brazilian vaccine strain, comparing it to that of BCG Pasteur. CFPs are considered of great importance given their dominant immunogenicity and role in pathogenesis, being available for interaction with host cells since early infection. Results The 2DE proteomic map of M. bovis BCG Moreau CFPs in the pH range 3 - 8 allowed the identification of 158 spots corresponding to 101 different proteins, identified by MS/MS. Comparison to BCG Pasteur highlights the great similarity between these BCG strains. However, quantitative analysis shows a higher expression of immunogenic proteins such as Rv1860 (BCG1896, Apa), Rv1926c (BCG1965c, Mpb63) and Rv1886c (BCG1923c, Ag85B) in BCG Moreau when compared to BCG Pasteur, while some heat shock proteins, such as Rv0440 (BCG0479, GroEL2) and Rv0350 (BCG0389, DnaK), show the opposite pattern. Conclusions Here we report the detailed 2DE profile of CFPs from M. bovis BCG Moreau and its comparison to BCG Pasteur, identifying differences that may provide relevant information on vaccine efficacy. These findings contribute to the detailed characterization of the Brazilian vaccine strain against TB, revealing aspects that may lead to a better understanding of the factors leading to BCG's variable protective efficacy against TB.

The influence of fish burger preparation and frying on residual levels of enrofloxacin (ENR) and ciprofloxacin (CIP) was evaluated. For this purpose, a high-throughput liquid chromatography-mass spectrometry analytical method for the quantitation of ENR and CIP residues in tilapia products (fillet, raw fish burger and fried fish burger) was developed and validated based on European and Brazilian guidelines. Sample preparation was accomplished by extraction with acidified acetonitrile followed by clean-up with hexane. Chromatographic analysis was performed on a C 18 column using isocratic elution with 0.1% formic acid and acetonitrile (85:15 v:v). The analytical method showed suitable performance to quantify the residual levels of ENR and CIP in the studied matrices. No reduction in the residual levels of ENR and CIP was observed during fish burger preparation and only a 10% reduction occurred as a consequence of frying, indicating that both compounds were stable to the preparation of the fish burger and to frying conditions.

Mononuclear cells have been implicated in the primary inflammatory response against mycobacteria. Yet, little is known about the interaction of Mycobacterium bovis bacillus Calmette-Guerin (BCG) with human monocytes. Here, we investigated the potential of BCG Moreau strain to induce in vitro specific cell-death utilizing a flow cytometry approach that revealed an increase in apoptosis events in BCG-stimulated monocytes from healthy adults. We also detected a concomitant release of interleukin 1 beta (IL-1β) and tumor necrosis factor alpha (TNF-α), but not metalloproteinase (MMP)-9. In addition, annexin V-propidium iodide double staining demonstrated an enhancement of monocytes necrosis, but not apoptosis, following BCG Moreau strain stimulation of umbilical vein cells from naïve, neonate. This pattern was paralleled by different pro-inflammatory cytokine levels, as well as MMP-9 induction when compared to the adults. Our findings support the hypothesis that BCG induces distinct cell-death patterns during the maturation of the immune system and that this pattern might set the stage for a subsequent antimycobacterial immune response that might have profound effects during vaccination., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The potent IFN-gamma inducing fusion antigen Ag85B-ESAT-6 (85B6) is a lead subunit candidate to improve current vaccination against Mycobacterium tuberculosis (Mtb). The recombinant M. bovis BCG strain Myc3504 was constructed to secrete 85B6. It was based on commercial BCG strain Moreau Rio de Janeiro (BCG(MoWT)) which remains available for human oral administration. Myc 3504 induced higher levels of 85B6-specific IFN-gamma circulating T-cells as compared to BCG(MoWT). A novel needle-free mucosal immunization regimen combining oral prime with Myc3504 or BCG(MoWT) with intranasal boost with LTK-63-adjuvanted 85B6 was compared to subcutaneous prime-boost immunization. Strikingly whereas parenteral immunization induced sustained levels of 85B6-specific IFN-gamma secretion by circulating T-cells, mucosal regimens induced barely detectable IFN-gamma. Despite this, mice and guinea pigs immunized with the mucosal regimens were as efficiently protected against aerosol Mtb challenge as parenterally immunized animals. After Mtb challenge, anti-ESAT-6 IFN-gamma responses sharply increased in non-vaccinated mice as a hallmark of infection. Parenterally immunized mice that controlled Mtb infection, displayed anti-ESAT-6 IFN-gamma responses as high as non-immunized infected mice, compromising the possible use of ESAT-6 as a diagnostic tool. Interestingly, in mucosally immunized mice that were equally protected, post-challenge ESAT-6-specific IFN-gamma T-cell response remained low.

The current tuberculosis (TB) vaccine Mycobacterium bovis BCG has been employed for some 70 years in Brazil and lessons from its use should be taken in account for the development or improvement of new TB vaccines. The vast majority of the current population has been vaccinated with BCG, with the possible requirement for a booster immunisation in adulthood for TB protection. BCG Moreau strain also protects against leprosy, meningitis and extrapulmonary forms of TB. Factors related to differences in strain, dosage and BCG administering protocol have been responsible for the variable efficacy of BCG. This vaccine is clearly affected by, as yet unclear, host and/or environmental variables. In this brief review, we describe some aspects of BCG immunisation observed in Brazil that may be of importance for improving or replacing BCG.

It has been proposed that antigens released by Trypanosoma cruzi sensitize vertebrate cells leading to their destruction by the immune response raised against the parasite. Here, we characterized antigens released by trypomastigotes of T. cruzi that bind to non-infected cells and investigated biological consequences of this adsorption. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of antigens released by [(35)S]-methionine-labeled parasites revealed the presence of polypeptides mainly ranging from 85 to 170 kDa that were specifically recognized by sera from chronically T. cruzi infected rabbits. Polypeptides of 85-110 and 160-170 kDa bound to non-infected epithelial, fibroblast and muscle mammalian cell lines, which thus became targets for anti-T. cruzi antibody binding. Cysteine-proteinase, but not trans-sialidase, was detected among the cell-bound antigens, and purified cysteine-proteinase was adsorbed to non-infected cells. Immunoelectron microscopic studies showed that parasite antigens were mainly released as membrane vesicles that adhered to membrane microvilli and were internalized by mammalian cells. We provide evidence that adsorption of parasite antigens induced an increase in expression of extracellular matrix (ECM) components (fibronectin, laminin and type I collagen) by sensitized cells. Thus, our data reinforce the idea that in vivo T. cruzi released antigens might be involved in the establishment of inflammation, sensitizing non-infected host cells and triggering an immune response against parasite antigens. Further, our data showed that antigen sensitization modulates biological cell functions as ECM expression that could mediate cell-cell or parasite-host cell interactions, contributing to the establishment of inflammation.

Three recombinant strains of Mycobacterium bovis Bacille Calmette Guerin (rBCG) were prepared in which the immunogenic B subunit of human Escherichia coli heat labile enterotoxin (LT-Bh) was expressed either as a cytoplasm protein, a cell wall associated lipoprotein or a secreted protein. Intraperitoneal immunisation of mice with these rBCG induced IgG and IgA antibodies to LT-Bh and shifted the serum IgG subclass response to subsequent challenge with purified LT-Bh from IgG1 to an IgG2a. Oral administration of recombinant BCG induced mucosal and serum IgA antibodies to LT-Bh which peaked four months after immunisation. Antibody responses were greater when LT-Bh was expressed as a secreted protein or lipoprotein rather than in the cytoplasm. Oral vaccination with recombinant BCG may be an effective approach, particularly to induce mucosal IgA and prime for a serum TH1 recall response.

Objective: Tuberculosis (TB) is the commonest HIV-related opportunistic infection in many developing countries and is thought to be a frequent underlying cause of HIV-associated wasting. We have used reference water dilution methods to examine the body composition changes associated with TB and to assess the severity and pattern of wasting., Methods: The study was conducted at a charitable support house for poor and homeless HIV-infected people in Rio de Janeiro, Brazil. Male patients who were HIV-positive and receiving treatment for active TB (HIVTB+) and HIV-infected controls without TB (HIVTB-) were studied. Total body water (TBW) and extracellular water (ECW) were measured by giving oral doses of deuterium oxide and sodium bromide, respectively, and determining enrichment in plasma after 4 hours. Intracellular water (ICW), body cell mass (BCM), lean body mass (LBM) and fat mass were calculated from these parameters using standard equations., Results: HIVTB+ (n = 11) and HIVTB- (n = 12) groups were similar in age, height, CD4 count and HIV risk factors. HIVTB+ men had significantly lower mean ICW (13.2 versus 16.6 kg; p = .02) and BCM (18.4 versus 23.0 kg; p = .02), a relative expansion of ECW (35.0 versus 30.0 L/kg body weight; p = .04), and small and nonsignificant reductions in total body weight (58.0 versus 62.1 kg; p = .26), LBM (45.5 versus 47.7 kg; p = .33) and fat mass (12.5 versus 14.4 kg; p = .51) compared with HIVTB- controls. BCM in the HIVTB+ group was similar to reference values for severe malnutrition. The relative depletion of BCM appeared excessive in comparison with reference values for uncomplicated starvation., Conclusion: The nutritional status of HIVTB+ patients was significantly worse than HIVTB- patients. Body weight and LBM underestimated the nutritional deficit, and measurement of BCM is therefore necessary to appreciate the extent of malnutrition in such patients. Malnutrition in HIVTB+ patients is severe and may therefore contribute to decreased survival. Hypermetabolism appears to play a role in the wasting process in patients coinfected with HIV and TB.

An indirect enzyme linked immunosorbent assay (ELISA) was applied to saliva to detect chronic infection by Trypanosoma cruzi in humans. Saliva samples from 114 Chagas' disease chronically infected individuals, characterized by three serological tests and clinical evaluation and from 100 healthy controls were tested for T. cruzi specific IgG antibodies. At dilution of 1 in 2, specific antibodies were detected in saliva samples from 103 of 114 samples from infected patients and 5 of 100 controls (sensitivity 90.4%, specificity 95%). There was no significant correlation between the antibody titre and cardiac or gastrointestinal tract disease. This assay possesses some advantages over other methods as saliva collection is non-invasive, requires no special equipment and whole saliva gave reproducible results. Although serology remains the gold standard for T. cruzi infection, these results suggest that T. cruzi specific salivary antibody detection may provide a screening diagnostic test and contribute to epidemiological studies of chronic trypanosomiasis infection in endemic areas.

Objective: Constant antigenic stimulation of the large immune cell population contained within gut-associated lymphoid tissue during HIV infection may contribute to patients' total viral load. The aim of this investigation was to evaluate the effect of a mucosal antigenic challenge on HIV replication., Design: Prospective clinical study., Methods: Twelve HIV-1-infected men (mean age, 42.3 years) from the Casa de Apoio Santo Antonio, Rio de Janeiro, Brazil, were immunized with combined whole cell-toxin B subunit oral cholera vaccine. Blood was collected on days 0, 2, 4, 6, 10 and 15 after immunization and plasma was tested for cholera toxin-specific antibody response (IgG and IgA), beta2-microglobulin, and plasma viral load. CD4 lymphocyte counts were performed within 1 week before immunization. Five HIV-infected non-immunized individuals were studied as controls., Results: There were no adverse effects following immunization and no deterioration in clinical outcome during 3 months of follow-up. A transient increase in viral load that ranged from twofold to 60-fold was observed in all cases and was statistically significant on days 2, 6 and 10 (P = 0.017, P = 0.025, P = 0.021, respectively). There was no correlation with CD4 cell counts. None of the non-immunized subjects demonstrated the pattern of viraemia observed after immunization (P > 0.10 on all days)., Conclusions: Our data indicate that mucosal immunization with oral cholera vaccine induces a transient increase in HIV viraemia, regardless of clinical stage of infection and CD4 cell counts. These findings suggest that mucosal stimulation of HIV-infected patients enhances HIV replication.

HIV-1-positive individuals were recruited from January 1993 to December 1996 from several cohorts receiving follow-up in the city of Rio de Janeiro, Brazil, to evaluate HIV-1 genetic variability and the potential association with modes of transmission. HIV-1 subtyping was carried out using the heteroduplex mobility assay (HMA), and those samples corresponding to the typical Brazilian subtype B variant were further identified based on the Fok I restriction fragment length polymorphism (RFLP). DNA sequencing was performed to evaluate one case of subtype D infection. From the 131 HIV-1-positive individuals analyzed, 106 (80.9%) could be identified as infected by subtype B and 20 (15.3%) by subtype F. One of the samples (0.8%) was classified as subtype D. DNA samples from 4 patients (3.0%) did not yield polymerase chain reaction (PCR)-amplified products to be typed. Based on the Fok I RFLP, 39 of the 106 subtype B samples (37%) were identified as corresponding to the typical Brazilian subtype B variant containing the GWGR motif at the tip of the V3 loop. No statistically significant association could be detected between HIV-I subtypes and modes of transmission, exposure categories, or gender. This is the first reported case of HIV-1 subtype D infection in Brazil.

Patients with secondary immunodeficiencies are at a high risk of infection. Currently some of these infections are preventable through specific immunization. Prevention of these diseases can diminish morbidity and mortality amongst these patients. In this review we describe the use of vaccines in persons with secondary immunodeficiencies.

Children born to HIV-1 infected mothers present a more severe clinical evolution than adults or children infected by other routes. The physiologic immaturity of the fetal and neonatal immune systems at the time of the infection probably plays an essential role in the progression of HIV-1 infection in these children. This paper describes the development of the normal human immune system and its correlation with the immunopathogenicity of vertical acquired immunodeficiency syndrome (AIDS).

The gut associated lymphoid tissue is responsible for specific responses to intestinal antigens. During HIV infection, mucosal immune deficiency may account for the gastrointestinal infections. In this review we describe the humoral and cellular mucosal immune responses in normal and HIV-infected subjects.

The mucosa associated lymphoid tissue regulates and coordinates immune responses against mucosal pathogens. Mucosal tissues are the major targets exposed to HIV during transmission. In this paper we describe in vitro models of HIV mucosal infection using human explants to investigate target cells within this tissue.

Objective: To determine the effect of HIV-1 infection on immunoglobulin (Ig) G and IgA antibody response and circulating antibody forming cell response to oral immunization with the B subunit of cholera toxin., Design: Healthy UK volunteers, and HIV-1-positive UK and Kenyan volunteers at different clinical stages of HIV-1 infection received two oral immunizations. CD4+ T cells, serum beta 2-microglobulin and neopterin were measured as surrogate markers of disease stage, and correlated with immunization response., Methods: Serum antitoxin IgG and IgA measured by enzyme-linked immunosorbent assay and antitoxin IgG, IgA and IgM antibody-forming cells detected by enzyme-linked immunospot assay at different times after two oral immunizations., Results: UK HIV-positive volunteers (mean CD4+ T cell count, 52 x 10(6)/l) responded poorly to primary and booster immunization. HIV-infected Kenyans (752 x 10(6)/l CD4+ T cells) had a significant primary and booster antibody response, whereas those with a mean CD4+ T cell count 186 x 10(6)/l had an insignificant primary, but significant booster response. Two oral immunizations induced antibody responses in HIV-positive Kenyan groups (who may have prior immunity from exposure to environmental bacterial toxins) of similar or greater magnitude to healthy UK volunteers., Conclusions: Mucosal immunization may recall immune memory and be of benefit in early and moderately advanced clinical HIV disease. The findings have important clinical implications in that mucosally targeted vaccines are potentially useful in this group of patients.

The kinetics and phenotypic characterization of the in vitro cell proliferative response to the B subunit of cholera toxin were studied using peripheral blood mononuclear cells taken from human volunteers at frequent time points after primary and booster oral immunizations. The cells induced to proliferate by oral immunization secreted IL-3, and lipopolysaccharide depletion and depletion of B cells did not affect proliferation. Flow cytometry demonstrated that activated cells were CD3- and CD4-positive. These findings indicate primed T cells proliferating specifically to the B subunit. The kinetics of the response suggested trafficking in the peripheral circulation of primed T cells from the gut, with a peak stimulation index of between 7 and 93 after first immunization, and a precursor frequency of primed cells of between 1 in 25,400 and 1 in 72,390. There was close correlation between the serum antitoxin IgA antibody levels and observed proliferation.

Peripheral blood mononuclear cells were taken from subjects before and after oral immunization with cholera toxin B-subunit. Cells obtained from naive volunteers before immunization did not proliferate in vitro to B-subunit. Oral immunization induced a proliferative response in all volunteers with a peak stimulation index of 20, and was detected up to 1 year later. The proliferative response kinetics suggest the appearance in the blood of primed T cells from the gut coinciding with the disappearance of primed plasmablasts from the circulation, supporting the concept of a common mucosal immune system in man for T and B cells.