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This meta-analysis of 13 studies revealed that amphotericin B delivered as a locally prepared lipid emulsion or in liposomes reduced nephrotoxicity to a similar degree, by 18.4% (relative risk [RR], 0.40 [99% confidence interval, .25-.64]; n = 459) and 18.1% (RR, 0.48 [99% CI, .36-.64]); n = 1233), respectively. more...

Infants born to HIV-infected mothers are at high risk of becoming infected during gestation or the breastfeeding period. A search is thus warranted for vaccine formulations that will prevent mother-to-child HIV transmission. The LAMP/gag DNA chimeric vaccine encodes the HIV-1 p55gag fused to the lysosome-associated membrane protein-1 (LAMP-1) and has been shown to enhance anti-Gag antibody (Ab) and cellular immune responses in adult and neonatal mice; such a vaccine represents a new concept in antigen presentation. In this study, we evaluated the effect of LAMP/gag DNA immunization on neonates either before conception or during pregnancy. LAMP/gag immunization of BALB/c mice before conception by the intradermal route led to the transfer of anti-Gag IgG1 Ab through the placenta and via breastfeeding. Furthermore, there were an increased percentage of CD4+CD25+Foxp3+T cells in the spleens of neonates. When offspring were immunized with LAMP/gag DNA, the anti-Gag Ab response and the Gag-specific IFN-γ-secreting cells were decreased. Inhibition of anti-Gag Ab production and cellular responses were not observed six months after immunization, indicating that maternal immunization did not interfere with the long-lasting memory response in offspring. Injection of purified IgG in conjunction with LAMP/gag DNA immunization decreased humoral and cytotoxic T-cell responses. LAMP/gag DNA immunization by intradermal injection prior to conception promoted the transfer of Ab, leading to a diminished response to Gag without interfering with the development of anti-Gag T- and B-cell memory. Finally, we assessed responses after one intravenous injection of LAMP/gag DNA during the last five days of pregnancy. The intravenous injection led to in utero immunization. In conclusion, DNA vaccine enconding LAMP-1 with Gag and other HIV-1 antigens should be considered in the development of a protective vaccine for the maternal/fetal and newborn periods. more...

We studied the induction of antigen-specific IgA memory B cells (B(M)) in volunteers who received live attenuated Shigella flexneri 2a vaccines. Subjects ingested a single oral dose of 10(7), 10(8) or 10(9) CFU of S. flexneri 2a with deletions in guaBA (CVD 1204) or in guaBA, set and sen (CVD 1208). Antigen-specific serum and stool antibody responses to LPS and Ipa B were measured on days 0, 7, 14, 28 and 42. IgA B(M) cells specific to LPS, Ipa B and total IgA were assessed on days 0 and 28. We show the induction of significant LPS-specific IgA B(M) cells in anti-LPS IgA seroresponders. Positive correlations were found between anti-LPS IgA B(M) cells and anti-LPS IgA in serum and stool; IgA B(M) cell responses to IpaB were also observed. These B(M) cell responses are likely play an important role in modulating the magnitude and longevity of the humoral response., (Copyright © 2011 Elsevier Inc. All rights reserved.) more...

Vaccines capable of inducing mucosal immunity in early postnatal life until adulthood, protecting early sexual initiation, should be considered as strategies to vaccination against HIV. The HIV-1 GAG protein as a chimera with the lysosome-associated membrane protein (LAMP/gag), encoded by a DNA vaccine, is targeted to the endosomal/lysosomal compartment that contains class II MHC molecules and has been shown to be immunogenic in adult mice. Assuming that one such strategy could help to overcome the immunological immaturity in the early postnatal period, we have evaluated the systemic and mucosal immunogenicity of LAMP/gag immunization in neonatal mice. Intranasal immunization with LAMP/gag vaccine induced higher levels of sIgA and IgG anti-GAG antibodies in intestinal washes than did the gag vaccine. The combination of ID injections and the IN protocol with the chimeric vaccine promoted the increase of Ab levels in sera. Both vaccines induced splenic IFN-γ- secreting cells against GAG peptide pools, as well as in vivo cytotoxic T lymphocyte (CTL) function, and increased the percentage of CD8+ T cells to the immunodominant class I peptide in gut and spleen. However, only the chimeric vaccine was able to enhance Th1/Th2 cytokine secretion in response to class II GAG peptide and to enhance IL-4-secreting cells against GAG peptides and p24 protein stimuli. Long-lasting humoral and cellular responses were detected until adult age, following neonatal immunization with the chimeric vaccine. The LAMP/gag vaccination was able to induce potent GAG-specific T and B cell immune responses in early life which are essential to elicit sustained and long-lasting mucosal and systemic humoral response., (Copyright © 2010 Elsevier GmbH. All rights reserved.) more...

Attenuated live oral typhoid vaccine candidate CVD 909 constitutively expresses Salmonella Typhi capsular polysaccharide antigen (Vi). A randomized, double-blind, heterologous prime-boost clinical study was conducted to determine whether immunity to licensed parenteral Vi vaccine could be enhanced by priming with CVD 909. Priming with CVD 909 elicited higher and persistent, albeit not significant, anti-Vi IgG and IgA following immunization with Vi, than placebo-primed recipients. Vi-specific IgA B memory (B(M)) cells were significantly increased in CVD 909-primed subjects. S. Typhi-specific LPS and flagella IgA B(M) cells were observed in subjects immunized with CVD 909 or with the licensed Vi-negative oral typhoid vaccine Ty21a. CVD 909-induced B(M) cells exhibited a classical B(M) phenotype (i.e., CD3(-)CD19(+)IgD(-)CD27(+)). This is the first demonstration of classical B(M) cells specific for bacterial polysaccharide or protein antigens following typhoid immunization. The persistent IgA B(M) responses demonstrate the capacity of oral typhoid vaccines to prime mucosally relevant immune memory., (Copyright © 2010 Elsevier Inc. All rights reserved.) more...

Epitopes from all available full-length sequences of yellow fever virus (YFV) and dengue fever virus (DENV) restricted by Human Leukocyte Antigen class I (HLA-I) alleles covering 12 HLA-I supertypes were predicted using the NetCTL algorithm. A subset of 179 predicted YFV and 158 predicted DENV epitopes were selected using the EpiSelect algorithm to allow for optimal coverage of viral strains. The selected predicted epitopes were synthesized and approximately 75% were found to bind the predicted restricting HLA molecule with an affinity, K(D), stronger than 500 nM. The immunogenicity of 25 HLA-A*02:01, 28 HLA-A*24:02 and 28 HLA-B*07:02 binding peptides was tested in three HLA-transgenic mice models and led to the identification of 17 HLA-A*02:01, 4 HLA-A*2402 and 4 HLA-B*07:02 immunogenic peptides. The immunogenic peptides bound HLA significantly stronger than the non-immunogenic peptides. All except one of the immunogenic peptides had K(D) below 100 nM and the peptides with K(D) below 5 nM were more likely to be immunogenic. In addition, all the immunogenic peptides that were identified as having a high functional avidity had K(D) below 20 nM. A*02:01 transgenic mice were also inoculated twice with the 17DD YFV vaccine strain. Three of the YFV A*02:01 restricted peptides activated T-cells from the infected mice in vitro. All three peptides that elicited responses had an HLA binding affinity of 2 nM or less. The results indicate the importance of the strength of HLA binding in shaping the immune response. more...

Background: The helminth Schistosoma mansoni parasite resides in mesenteric veins where fecundated female worms lay hundred of eggs daily. Some of the egg antigens are trapped in the liver and induce a vigorous granulomatous response. High Mobility Group Box 1 (HMGB1), a nuclear factor, can also be secreted and act as a cytokine. Schistosome HMGB1 (SmHMGB1) is secreted by the eggs and stimulate the production of key cytokines involved in the pathology of schistosomiasis. Thus, understanding the mechanism of SmHMGB1 release becomes mandatory. Here, we addressed the question of how the nuclear SmHMGB1 can reach the extracellular space., Principal Findings: We showed in vitro and in vivo that CK2 phosphorylation was involved in the nucleocytoplasmic shuttling of SmHMGB1. By site-directed mutagenesis we mapped the two serine residues of SmHMGB1 that were phosphorylated by CK2. By DNA bending and supercoiling assays we showed that CK2 phosphorylation of SmHMGB1 had no effect in the DNA binding activities of the protein. We showed by electron microscopy, as well as by cell transfection and fluorescence microscopy that SmHMGB1 was present in the nucleus and cytoplasm of adult schistosomes and mammalian cells. In addition, we showed that treatments of the cells with either a phosphatase or a CK2 inhibitor were able to enhance or block, respectively, the cellular traffic of SmHMGB1. Importantly, we showed by confocal microscopy and biochemically that SmHMGB1 is significantly secreted by S. mansoni eggs of infected animals and that SmHMGB1 that were localized in the periovular schistosomotic granuloma were phosphorylated., Conclusions: We showed that secretion of SmHMGB1 is regulated by phosphorylation. Moreover, our results suggest that egg-secreted SmHMGB1 may represent a new egg antigen. Therefore, the identification of drugs that specifically target phosphorylation of SmHMGB1 might block its secretion and interfere with the pathogenesis of schistosomiasis. more...

Embryonic stem (ES) cells are pluripotent cells derived from the inner cell mass of blastocyst-stage early mammalian embryos. A crucial stage in the differentiation of ES cells is the formation of embryoid bodies (EBs) aggregates. EB formation is based on spontaneous aggregation when ES cells are cultured in non adherent plates. Three-dimensional EB recapitulates many aspects of early mammalian embryogenesis and differentiate into the three germ layers: ectoderm, mesoderm and endoderm. Immunofluorescence and in situ hybridization are widely used techniques for the detection of target proteins and mRNA present in cells of a tissue section. Here we present a simple technique to generate high quality cryosections of embryoid bodies. This approach relies on the spatial orientation of EB embedding in OCT followed by the cryosection technique. The resulting sections can be subjected to a wide variety of analytical procedures in order to characterize populations of cells containing certain proteins, RNA or DNA. In this sense, the preparation of EB cryosections (10 μm) are essential tools for histology staining analysis (e.g. Hematoxilin and Eosin, DAPI), immunofluorescence (e.g. Oct4, nestin) or in situ hybridization. This technique can also help to understand aspects of embryogenesis with regards to the maintenance of the tri-dimensional spherical structure of EBs. more...

Successful T cell priming in early postnatal life that can generate effective long-lasting responses until adulthood is critical in HIV vaccination strategies because it prevents early sexual initiation and breastfeeding transmission of HIV. A chimeric DNA vaccine encoding p55 HIV gag associated with lysosome-associated membrane protein 1 (LAMP-1; which drives the antigen to the MIIC compartment), has been used to enhance cellular and humoral antigen-specific responses in adult mice and macaques. Herein, we investigated LAMP-1/gag vaccine immunogenicity in the neonatal period in mice and its ability to generate long-lasting effects. Neonatal vaccination with chimeric LAMP/gag generated stronger Gag-specific immune responses, as measured by the breadth of the Gag peptide-specific IFN-gamma, proliferative responsiveness, cytokine production and antibody production, all of which revealed activation of CD4+ T cells as well as the generation of a more robust CTL response compared to gag vaccine alone. To induce long-lived T and B cell memory responses, it was necessary to immunize neonates with the chimeric LAMP/gag DNA vaccine. The LAMP/gag DNA vaccine strategy could be particularly useful for generating an anti-HIV immune response in the early postnatal period capable of inducing long-term immunological memory., (Copyright © 2010 Elsevier Inc. All rights reserved.) more...

Although researchers have related sexual desire in older women to quality-of-life variables such as overall physical health, well-being, and life satisfaction, little is known about the socio-cultural mechanisms that shape sexual desire in minority ethnic older women. We investigated this sexual variable among Mexican-American older women in a qualitative fashion. Data were collected from 25 community-dwelling women of Mexican descent (aged 59-89 years) using a semi-structured interview protocol and a grounded theory approach. We inquired about dimensions of sexual desire including sexual fantasies and the desire to engage in sexual activity within the context of several socio-cultural and health-related factors. Using content analysis, we were able to identify key themes differentiating among respondents' levels of sexual desire and fantasies. These included the availability of a suitable partner, cultural and religious norms pertinent to women's sexuality, stigma related to sexuality in older age, and health status. Traditional socio-cultural restrictions coupled with unmarried status and physical health problems emerged as critical issues associated with limited or no sexual fantasies and desire in our sample. Many respondents indicated that their sexual needs were unmet. more...

Lipid peroxidation (LP) is one of the most harmful mechanisms developed after spinal cord (SC) injury. Several strategies have been explored in order to control this phenomenon. Protective autoimmunity is a physiological process based on the modulation of inflammatory cells that can be boosted by immunizing with neural-derived peptides, such as A91. Since inflammatory cells are among the main contributors to lipid peroxidation, we hypothesized that protective autoimmunity could reduce LP after SC injury. In order to test this hypothesis, we designed two experiments in SC contused rats. First, animals were immunized with a neural-derived peptide seven days before injury. With the aim of inducing the functional elimination of CNS-specific T cells, for the second experiment, animals were tolerized against SC-protein extract and thereafter subjected to a SC injury. The lipid-soluble fluorescent products were used as an index of lipid peroxidation and were assessed after injury. Immunization with neural-derived peptides reduced lipid peroxidation after SC injury. Functional elimination of CNS-specific T cells avoided the beneficial effect induced by protective autoimmunity. The present study demonstrates the beneficial effect of immunizing with neural-derived peptides on lipid peroxidation inhibition; besides this, it also provides evidence on the neuroprotective mechanisms exerted by protective autoimmunity., (Copyright 2010 Elsevier Ireland Ltd. All rights reserved.) more...

Shigella dysenteriae type 1 can cause devastating pandemics with high case fatality rates; a vaccine for Shigella is unavailable currently. Because of the risks associated with performing challenge studies with wild-type S. dysenteriae 1 in human clinical trials to advance vaccine development, an improved nonhuman primate model is needed urgently. In the present study, cynomolgus macaques (Macaca fascicularis) were challenged with various doses of S. dysenteriae 1 strain 1617 to establish a dose that would produce shigellosis. Further, different routes of delivery of S. dysenteriae 1 were compared to establish the most appropriate route for infection. Animals receiving 10(11) cfu S. dysenteriae 1 intragastrically consistently developed signs of shigellosis characterized by the onset of diarrhea and dysentery within 2 to 3 d. Administration of as many as 10(9) cfu S. dysenteriae 1 intraduodenally did not elicit signs characteristic of infection in macaques despite fecal shedding of bacteria for as long as 10 d. S. dysenteriae 1 administered intraduodenally at 10(9) cfu or intragastrically at 10(11) cfu elicited robust IgG and IgA antibody responses to LPS. We have developed a reliable challenge model of infection with wild-type S. dysenteriae 1 in cynomolgus macaques that reproducibly induces disease and elicits robust immune responses. We believe that this animal model may provide unique insights into the immunologic mechanisms of protection to S. dysenteriae 1 infection and in advancing development of a vaccine against shigellosis. more...

The heterogeneous acid-catalyzed activation step of alkanes leading to the reaction intermediates (carbocationic or alkoxy species) was up to now the matter of a longstanding controversy. Gas chromatography and online mass spectroscopy measurements show that H(2) and methane are formed over H-zeolites, whereas HD and CH(3)D are formed over D-zeolites as the primary products in the reaction with isobutane. These results indicate that sigma-bond protolysis by strong acid sites is the first step for hydrocarbon activation on these catalysts at mild temperatures (473 K), in analogy to the activation path occurring in liquid superacid media. more...

Vaccination is the most practical and cost-effective strategy to prevent the majority of the flavivirus infection to which there is an available vaccine. However, vaccines based on attenuated virus can potentially promote collateral side effects and even rare fatal reactions. Given this scenario, the development of alternative vaccination strategies such as DNA-based vaccines encoding specific flavivirus sequences are being considered. Endogenous cytoplasmic antigens, characteristically plasmid DNA-vaccine encoded, are mainly presented to the immune system through Major Histocompatibility Complex class I - MHC I molecules. The MHC I presentation via is mostly associated with a cellular cytotoxic response and often do not elicit a satisfactory humoral response. One of the main strategies to target DNA-encoded antigens to the MHC II compartment is expressing the antigen within the Lysosome-Associated Membrane Protein (LAMP). The flavivirus envelope protein is recognized as the major virus surface protein and the main target for neutralizing antibodies. Different groups have demonstrated that co-expression of flavivirus membrane and envelope proteins in mammalian cells, fused with the carboxyl-terminal of LAMP, is able to induce satisfactory levels of neutralizing antibodies. Here we reviewed the use of the envelope flavivirus protein co-expression strategy as LAMP chimeras with the aim of developing DNA vaccines for dengue, West Nile and yellow fever viruses. more...

Optimized DNA expression vectors encoding the native HIV-1 Gag or a fusion of Gag with the lysosomal membrane associated protein 1 (LAMP) were compared for immunogenicity upon intramuscular DNA delivery in rhesus macaques. Both vaccines elicited CD4(+) T-cell responses, but with significant differences in the phenotype of the Gag-specific cells: the native Gag induced CD4(+) responses with a phenotype of central memory-like T cells (CD28(+) CD45RA(-)), whereas the LAMP/Gag chimera induced CD4(+) responses with effector memory phenotype (CD28(-) CD45RA(-)). Antigen-specific T cells producing both IFN-gamma and TNFalpha were found in the animals receiving the native Gag, whereas the LAMP/Gag chimera induced humoral responses faster. These results demonstrate that modification of intracellular Gag trafficking results in the induction of distinct immune responses. Combinations of DNA vectors encoding both forms of antigen may be more potent in eliciting anti-HIV-1 immunity. more...

We evaluated B memory responses in healthy adult volunteers who received one oral dose of live-attenuated Shigella flexneri 2a vaccine. LPS-specific B(M) cells increased from a median of 0 at baseline to 20 spot forming cells (SFC)/10(6) expanded cells following vaccination (p=0.008). A strong correlation was found between post-vaccination anti-LPS B(M) cell counts and peak serum anti-LPS IgG titers (rs=0.95, p=0.0003). Increases in B(M) specific for IpaB approaching significance were also observed. In sum, oral vaccination with live-attenuated S. flexneri 2a elicits B(M) cells to LPS and IpaB, suggesting that B(M) responses to Shigella antigens should be further studied as a suitable surrogate of protection in shigellosis. more...

Immunomics research uses in silico epitope prediction, as well as in vivo and in vitro approaches. We inoculated BALB/c (H2d) mice with 17DD yellow fever vaccine to investigate the correlations between approaches used for epitope discovery: ELISPOT assays, binding assays, and prediction software. Our results showed a good agreement between ELISPOT and binding assays, which seemed to correlate with the protein immunogenicity. PREDBALB/c prediction software partially agreed with the ELISPOT and binding assay results, but presented low specificity. The use of prediction software to exclude peptides containing no epitopes, followed by high throughput screening of the remaining peptides by ELISPOT, and the use of MHC-biding assays to characterize the MHC restrictions demonstrated to be an efficient strategy. The results allowed the characterization of 2 MHC class I and 17 class II epitopes in the envelope protein of the YF virus in BALB/c (H2d) mice. more...

Previous studies of HIV-1 p55Gag immunization of mice have demonstrated the usefulness of targeting antigens to the cellular compartment containing the major histocompatibility complex type II (MHC II) complex molecules by use of a DNA antigen formulation encoding Gag as a chimera with the mouse lysosome-associated membrane protein (mLAMP/gag). In the present study, we have analyzed the magnitude and breadth of Gag-specific T-lymphocyte and antibody responses elicited in Rhesus macaques after immunization with DNA encoding a human LAMP/gag (hLAMP/gag) chimera. ELISPOT analyses indicated that the average Gag-specific IFN-gamma response elicited by the hLAMP/gag chimera was detectable after only two or three naked DNA immunizations in all five immunized macaques and reached an average of 1000 spot-forming cells (SFC)/10(6) PBMCs. High IFN-gamma ELISPOT responses were detected in CD8(+)-depleted cells, indicating that CD4(+) T-cells play a major role in these responses. The T-cell responses of four of the macaques were also tested by use of ELISPOT to 12 overlapping 15-amino acids (aa) peptide pools containing ten peptides each, encompassing the complete Gag protein sequence. The two Mamu 08 immunized macaques responded to eight and twelve of the pools, the Mamu B01 to six, and the other macaque to five pools indicating that the hLAMP/gag DNA antigen formulation elicits a broad T-cell response against Gag. Additionally, there was a strong HIV-1-specific IgG response. The IgG antibody titers increased after each DNA injection, indicating a strong amnestic B-cell response, and were highly elevated in all the macaques after three immunizations. Moreover, the serum of each macaque recognized 13 of the 49 peptides of a 20-aa peptide library covering the complete Gag amino acid sequence. In addition, HIV-1-specific IgA antibodies were present in the plasma and external secretions, including nasal washes. These data support the findings of increased immunogenicity of genetic vaccines encoded as LAMP chimeras, including the response to DNA vaccines by non-human primates. more...

Ag processing is a critical step in defining the repertoire of epitope-specific immune responses. In the present study, HIV-1 p55Gag Ag was synthesized as a DNA plasmid with either lysosomal-associated membrane protein-1 (LAMP/gag) or human dendritic cell-LAMP (DC-LAMP/gag) and used to immunize mice. Analysis of the cellular trafficking of these two chimeras demonstrated that both molecules colocalized with MHC class II molecules but differed in their overall trafficking to endosomal/lysosomal compartments. Following DNA immunization, both chimeras elicited potent Gag-specific T and B cell immune responses in mice but differ markedly in their IL-4 and IgG1/IgG2a responses. The DC-LAMP chimera induced a stronger Th type 1 response. ELISPOT analysis of T cell responses to 122 individual peptides encompassing the entire p55gag sequence (15-aa peptides overlapping by 11 residues) showed that DNA immunization with native gag, LAMP/gag, or DC-LAMP/gag induced responses to identical immunodominant CD4+ and CD8+ peptides. However, LAMP/gag and DC-LAMP/gag plasmids also elicited significant responses to 23 additional cryptic epitopes that were not recognized after immunization with native gag DNA. The three plasmids induced T cell responses to a total of 39 distinct peptide sequences, 13 of which were induced by all three DNA constructs. Individually, DC-LAMP/gag elicited the most diverse response, with a specific T cell response against 35 peptides. In addition, immunization with LAMP/gag and DC-LAMP/gag chimeras also promoted Ab secretion to an increased number of epitopes. These data indicate that LAMP-1 and DC-LAMP Ag chimeras follow different trafficking pathways, induce distinct modulatory immune responses, and are able to present cryptic epitopes. more...

Niemann Pick type C is a severe, incurable disease caused, in the majority of cases, by mutations in the Niemann Pick type C protein 1 (NPC1). The pathology and biochemical changes associated with the disease have been extensively studied. However, the function of the protein is still unknown, and recent studies challenge the established concept that a defect in cholesterol and sphingolipids transport is the primary cause of this human lipidosis. Clearly defining the mechanisms by which defects in this protein lead to the disease phenotype will require further studies on the structure and function of this protein. Therefore, the development of a well-characterized monoclonal antibody (MAb) against this protein to facilitate such studies is an important goal. Here, we describe the production and characterization of such a MAb. The antibody is demonstrated to be highly specific and species cross-reactive. Function studies show that the antibody induces the NPC1 disease phenotype in cells, making it highly likely that the antibody blocks function of the NPC1 protein. more...

Durable antigen (Ag)-specific T- and B-cell anergy induced by oral tolerance is an attractive strategy for immunotherapy of allergic diseases. Here, we address the lasting effect of oral tolerance induction in naïve or primed mice to ovalbumin (OVA) on antibody production. Single feeding with OVA prior to immunization or double feeding, before and after Ag priming, in A/Sn mice, induced a long-lasting suppression of IgE, IgG1 and IgG2a responses up to 8 months after immunization. In contrast, primed-fed mice had transient IgE inhibition. Naive and double-treated mice showed marked Ag-specific unresponsiveness and scarce cytokines production. Inhibition of IL-2 and IFN-gamma secretion in naïve-fed mice were restored in the presence of anti-CD28 mAb plus Ag stimulation. The durable inhibition of Ab production in OVA-fed mice was related to the persistent decrease of B7.2 expression on B cells. Ag feeding in naive and primed status may be a prophylactic measure to avoid later Ag sensitization. more...

Induced oral tolerance to mucosal-exposed antigens in immunized animals is of particular interest for the development of immunotherapeutic approaches to human allergic diseases. This is a unique feature of mucosal surfaces which represent the main contact interface with the external environment. However, the influence of oral tolerance on specific and natural polyreactive IgA antibodies, the major defense mechanism of the mucosa, is unknown. We have shown that oral administration of an extract of the dust mite Dermatophagoides pteronyssinus (Dp) to primed mice caused down-regulation of IgE responses and an increase in tumor growth factor-beta secretion. In the present study, we observed that primed inbred female A/Sn mice (8 to 10 weeks old) fed by gavage a total weight of 1.0-mg Dp extract on the 6th, 7th and 8th days post-immunization presented normal secretion of IL-4 and IL-10 in gut-associated lymphoid tissue and a decreased production of interferon gamma induced by Dp in the draining lymph nodes (13,340 +/- 3,519 vs 29,280 +/- 2,971 pg/ml). Mice fed the Dp extract also showed higher levels of serum anti-Dp IgA antibodies and an increase of IgA-secreting cells in mesenteric lymph nodes (N = 10), reflecting an increase in total fecal IgA antibodies (N = 10). The levels of secretory anti-Dp IgA antibodies increased after re-immunization regardless of Dp extract feeding. Oral tolerance did not interfere with serum or secretory IgA antibody reactivity related to self and non-self antigens. These results suggest that induction of oral tolerance to a Dp extract in sensitized mice triggered different regulatory mechanisms which inhibited the IgE response and stimulated systemic and secretory IgA responses, preserving the natural polyreactive IgA antibody production. more...

Two experiments were conducted to evaluate dry matter intake (DMI), milk yield, and milk composition from feeding rations that contained different sources of genetically modified whole cottonseed to Argentinean Holstein dairy cows. Twenty-four lactating multiparous Argentinean Holstein dairy cows were used in 2 experiments with a replicated 4 x 4 Latin square design, with cows averaging 565 kg body weight and 53 d in milk at the beginning of the experiments. Treatments in Experiment 1 were: Bollgard cotton containing the cry1Ac gene, Bollgard II cotton containing cry1Ac and cry2Ab genes, Roundup Ready cotton containing the cp4 epsps gene, and a control nongenetically modified but genetically similar cottonseed. In Experiment 2, two commercial sources, a parental control line, and the transgenic cotton containing both cry1Ac and cp4 epsps genes were used as treatments. All cows received the same total mixed ration but with different whole cottonseed sources. Cottonseed was included to provide 2.50 kg per cow daily (dry matter [DM] basis) or about 10% of the total diet DM. The ingredient composition of the total mixed ration was 32% alfalfa hay, 28% corn silage, 22% corn grain, 17% soybean meal, and 2% minerals and vitamins. In addition, genomic DNA was extracted from a subset of milk samples and analyzed by polymerase chain reaction followed by Southern blot hybridization for small fragments of the cry1Ac transgene and an endogenous cotton gene, acp1. No sample was positive for transgenic or plant DNA fragments at the limits of detection for the assays following detailed data evaluation criteria. The DMI, milk yield, milk composition, body weight, and body condition score did not differ among treatments. Cottonseed from genetically modified varieties used in these studies yielded similar performance in lactating dairy cows when compared to non-transgenic control and reference cottonseed. more...

A rare case of isolated sphenoid sinus aspergillosis in a 51-year-old patient, with the clinical and laboratory diagnosis of Cushing's disease but no radiologically confirmed tumor is presented. The patient made a complete recovery after treatment. The radiologic findings and the management of this potential disease are discussed. more...

Background: The dust mites Dermatophagoides pteronyssinus (Dp) and Blomia tropicalis (Bt) are important sources of indoor allergens in tropical and subtropical countries. Murine models allow the analysis of the immune response and regulation of IgE production to Dp and Bt allergens. Oral tolerance induces unresponsiveness in naive animals, but its application in sensitized animals can provide useful information to improve allergy therapy., Objective: To study the profile of IgE and IgG subclasses antibody upon oral administration with Bt and Dp extract in previously sensitized mice. Further, the occurrence of autoantibodies IgG anti-IgE in the immunization and in the oral tolerance was investigated., Methods: A/Sn mice were immunized with Bt or Dp extract in alum, orally administrated with 0.25 mg of Bt or Dp extract or PBS at the 6th, 7th and 8th days after immunization and boosted twice with their respective allergens. To analyse the mice groups, specific IgE antibodies were measured by passive anaphylaxis reaction and specific IgG subclasses and anti-IgE IgG autoantibody by ELISA assay., Results: IgE levels were markedly increased in Bt-immunized mice compared with Dp-immunized mice. A distinct profile of the specific isotypes was verified in Bt-immunized mice with a preferential production of IgG3 and IgA antibodies, whereas Dp-immunized mice developed high titres of anti-Dp IgG1, IgG2a and IgG2b antibodies. The antigen feeding inhibited IgE response in both fed-mice groups but only Dp-fed mice presented decreased levels of IgG antibodies. Free anti-IgE IgG autoantibodies were detected mainly in the Dp-immunization and they correlated with the antibody isotypes found against the allergen., Conclusions: This is the first time that the murine-type I hypersensitivity is employed to study Bt-immunization, showing a marked IgE production, associated with IgG response, which is at least in part driven by T-independent antigens. The oral tolerance protocol in previously sensitized animals was able to down-modulate IgE response and points out this route as a strategy for allergy therapy. more...

Background: Maternal exposure to environmental ubiquitous allergens could exert an influence on the newborn's immune repertoire and the later development of allergy. The aim of this study was to investigate the effects of maternal immunization with Dermatophagoides pteronyssinus (Dp) on the hypersensitivity response and IgG subclass production in offspring using a murine model., Methods: A/Sn mice were immunized with Dp before mating with normal A/Sn males. Diaplacental serum samples were collected from newborn mice delivered by cesarean section, and maternal milk samples were extracted from the stomachs of newborn mice. Groups of offspring 25 or 45 days old were Dp immunized and boosted on the 10th day after sensitization. The animals were bled 7 days after the booster., Results: High levels of anti-Dp IgG subclasses - mainly IgG1, but also IgG2a and IgG2b - were transmitted by immunized mice via the placenta to the offspring. In the milk from immunized mothers, significant levels of anti-Dp IgG subclasses and anti-Dp IgM and IgA antibodies were detected. Moreover, the increase in total IgA antibodies in the milk of the immunized females correlated with a significantly increased level of TGF-beta1. TGF-beta2 levels were markedly higher than the beta1 isoform in the milk, although no difference was observed between the groups. When offspring from immunized mothers were sensitized at 25 days, a significant decrease in total and anti-Dp IgE antibodies as well as total and anti-Dp IgG1, IgG2a and IgG2b subclasses was observed compared to normal female offspring, whereas when offspring were sensitized at 45 days, both offspring groups showed similar levels of IgE and IgG subclasses., Conclusions: Our study showed that maternal immunization with Dp promotes the transference of specific antibodies and/or TGF-beta, which can negatively modulate the allergic response in offspring, and suggests that maternal preexposure to allergen before mating can protect mice during the early phase., (Copyright 2002 S. Karger AG, Basel) more...

Oral antigen administration induces peripheral tolerance in naive animals. Studies of oral tolerance induction in sensitized mice have clinical relevance as a strategy to modulate allergy. In this study, the A/Sn mice sensitized with extract of Dermatophagoides pteronyssinus (Dp) and submitted to oral Dp administration showed a marked decrease in IgE anti-Dp antibody production compared with sensitized phosphate-buffered saline (PBS)-fed mice. T cells from Dp-fed mice cocultured with spleen cells from PBS-fed mice were able to inhibit IgE anti-Dp antibody production and did not interfere in IgG1 antibody levels. The analysis of cytokine profile after Dp feeding showed a significant decrease in interleukin-4 (IL-4), IL-5, and IL-13 antigen-induced secretion levels by spleen cells, without shifting to IL-2 and interferon-gamma (IFN-gamma) production. Both transforming growth factor-beta (TGF-beta) baseline and TGF-beta antigen-stimulated levels were increased in Dp-fed mice. The effects of regulatory cytokines on anti-Dp IgE antibody production were investigated in vitro. The addition of recombinant TGF-beta (rTGF-beta) to spleen cell cultures stimulated by Dp inhibited IgE antibody secretion in both mouse groups. Neutralizing antibodies to IL-4, but not anti-TGF-beta, induced a marked inhibition of IgE production. Therefore, a negative modulatory effect on IgE response by inhibition of the axis Th2 was observed in sensitized Dp-fed mice, possibly mediated by induction of regulatory cytokines. more...

Such allergic diseases as rhinitis and asthma are IgE-mediated type I reactions and are controlled primarily by Th2 cells. One of the major dust mites, Dermatophagoides pteronyssinus (Dp), is considered to cause allergic reactions. Oral tolerance, largely used to modulate immune response, opens the possibility of modulating Th2 allergic responses. We observed downmodulation of total and specific IgE antibody levels as well as the number of specific IgE-secreting cells with Dp feeding in previously sensitized mice. Analysis of the cytokine profile in mucosal lymphoid tissues in the protocol revealed altered patterns of interferon-gamma (IFN-gamma), interleukin-5 (IL-5), and transforming growth factor-beta (TGF-beta) secretion in Dp-fed animals. The results suggest that both the Th and B cell populations are modulated in mice made tolerant by oral Dp feeding. Understanding the mechanisms at the mucosal level that underlie oral tolerance can improve its use in allergy immunotherapy. more...

One of the main goals of immunotherapy of allergic diseases is the down-regulation of the type I hypersensitivity reaction. We investigated in this study the effect of oral administration of varying doses (0.25, 1.0, 4.0 and 10 mg) of dust mite extract (Dermatophagoides pteronyssinus, Dp) in sensitized A/Sn mice. A marked decrease of the allergen-specific immunoglobulin E (IgE) response was observed with all antigen doses. The mice orally tolerized with low Dp dose (0.25 mg) had a significant decrease in the total serum IgE and in the immunoglobulin G1 (IgG1), IgG2a and IgG2b antibody levels. The higher Dp dose (10.0 mg), however, enhanced the IgG1 antibody response, suggesting the stimulation of a pre-existing immune response of the sensitized animals. Animals fed with the low Dp dose had a significant decrease in the frequency of interleukin-4 (IL-4) secreting cells. These animals also showed a significant decrease in the frequency of Dp-specific IgE- and IgG1-positive plasma cells. Our data suggest that feeding dust mite extract to Dp-sensitized mice down-regulates the development of type I hypersensitivity, by inhibition of the T helper 2 response. more...

Objective: Although improving mothers' knowledge about breastfeeding can increase rates and duration of breastfeeding, little is known about the influence of fathers' knowledge. The purpose of this study was to assess the knowledge of mothers and fathers about breastfeeding before and after receiving postpartum advice and its relationship to the frequency of breastfeeding., Methods: A clinical trial was performed with mothers and fathers of normal children born at the Hospital de Clínicas de Porto Alegre, Brazil, between July 1994 and March 1995. The study intervention consisted of postpartum advice supplied by means of a video film discussing basic topics of breastfeeding, an explanatory leaflet, and open discussion after viewing the video. The first 208 couples comprised the control group, the next 197 comprised experimental group 1, and the remaining 196 comprised experimental group 2. Immediately after delivery, mothers and fathers in the three groups answered a test on breastfeeding knowledge; they completed the same test at the end of the first month. All families received home visits at the end of the first, second, fourth, and sixth months, or until breastfeeding ceased. Logistic regression was used to evaluate the association between the mothers' and fathers' knowledge and frequency of breastfeeding., Results: Postpartum advice increased the breastfeeding knowledge of mothers and fathers. The mothers with the highest level of knowledge had a 6.5 times higher chance of exclusively breastfeeding at the end of the third month, and 1.97 times higher chance of continuing breastfeeding to the end of the sixth month compared with other mothers. The fathers' knowledge also significantly influenced breastfeeding rates. The children whose fathers knew more had a 1.76 higher chance of being exclusively breastfed at the end of the first month, and 1.91 higher chance of receiving maternal milk at the end of the third month., Conclusion: A simple, inexpensive strategy can increase the level of breastfeeding knowledge of mothers and fathers and, consequently, have a positive impact on the frequency of breastfeeding. more...

We investigated the effect on specific antibody response of naive and sensitized mice orally administrated with low (0.25 mg) or high (10.0 mg) doses of Dermatophagoides pteronyssinus (Dp) extract. We also examined the effect of oral administration of Dp on the production of autoantibodies to immunoglobulin G (IgG) and immunoglobulin E (IgE). Naive and sensitized mice both showed a marked down-regulation of IgE antibody production, regardless of the dose of Dp. We also detected an inhibitory effect of the total IgE levels and the allergen-specific IgG1, IgG2a and IgG2b antibody response in sensitized mice given the low dose of Dp. In contrast, high doses of Dp stimulated IgG1 antibody production in both naive and sensitized animals. In addition, the oral tolerance induction protocol stimulated anti-F(ab')2gamma and anti-Fcgamma autoantibody production. Evaluation of IgG anti-IgE autoantibodies by a direct enzyme immunoassay (EIA) revealed the presence of these autoantibodies, predominantly of the IgG1 isotype, specifically in those animals fed with the high dose. In contrast, IgG-IgE complexes, determined by EIA using immobilized anti-IgE antibodies, were detected mainly in sera of control animals. The autoantibody anti-IgE specificity was tested against IgE-TNP and IgE-DANSYL murine proteins and revealed different inhibition profiles, suggesting the action of heterogeneous subpopulations of autoantibodies. Taken together, our results show that the oral tolerance protocol with Dp was able to modulate the production of allergen-specific IgE antibodies in both naive and sensitized animals. In addition, we suggest that anti-IgE autoantibodies participate in the modulation of allergic response triggered by oral tolerance protocols. more...

OBJECTIVE: To assess mother's knowledge of breastfeeding before and after guidance supplied during the postpartum period and its relationship to the prevalence of breastfeeding.METHODS: A clinical trial was performed with 405 mothers of normal children born at Hospital de Clínicas de Porto Alegre from July to December 1994. The intervention consisted of guidance given by means of a video film discussing basic topics on breastfeeding, an explanatory leaflet and open discussion after the video. The first 208 mothers constituted the Control Group and the remaining 197 the Experimental Group. All mothers answered a question form for identification purposes and a test on previous knowledge regarding breastfeeding in the maternity ward. The mothers in both groups were followed by means of home visits at the end of the first, second, fourth and sixth months, or until they stopped breastfeeding. At the end of the first month the mothers were submitted to the same test given right after delivery. Logistic regression was used to evaluate the association between the motherś knowledge of breastfeeding and the prevalence of breastfeeding.RESULTS: The mothers who received the intervention (Experimental Group) had a significantly higher score in the tests on knowledge of breastfeeding at the end of the first month as compared with the mothers in the Control Group (17.0 versus 14.7). The intervention increased by 1.7 mothers chances of achieving a score above the average. The mothers whose scores were above the average had a 8.2 higher chance of being breastfeeding exclusively at the end of the third month and twice as high of still being breastfeeding at the end of the sixth month.CONCLUSION: Simple strategies to increase mother's knowledge regarding breastfeeding can have a positive impact on breastfeeding rates. more...

The present study describes the antimicrobiological methods used for ooscopic instruments and also recommends a routine of material caring, methods and products to be employed. These orientations were also based on the author's experience with those methods of cleaning, disinfection and sterilization at a school hospital. It is expected to simplify the procedures describing its steps with scientific embasement. more...

To determine the influence of a dominant follicle on the superovulatory response in midlate lactating cows, the dynamics of follicular growth and the development of corpora lutea and embryos were studied. Real-time B-mode ultrasonography with a 7.5 MHz rectal linear-array transducer was used to scan the ovaries daily, from 8-12 days before superovulation until day 7 post-estrus, when embryos were recovered. On the first day of superovulation cows were classified as belonging to either a 'dominant' group (n = 12) having a 'dominant follicle', i.e. > 9 mm in diameter in growing phase or stable for < 4 days, or a 'non-dominant' group (n = 11) those carrying a 'non-dominant follicle', i.e. a follicle in a regressing phase or with the same diameter for > 4 days. Cows in the 'dominant' group had significantly higher numbers of 4-6 and 7-10 mm follicles than cows in the 'non-dominant' group on the third and the fifth day of superovulation. The total number of follicles (> 4 mm) preovulation and the number of CL day 7 postovulation was higher in the 'dominant' group than in the 'non-dominant' group. There were no differences between groups regarding the total number of embryos or transferable embryos recovered. The results indicate that superovulation in mid-late lactation is not negatively affected by the presence of a 'dominant follicle'. The criteria to define 'dominant' and 'non-dominant' follicles might be redefined when applied to lactating cows, whose response to superovulation seems to be under the influence of many other factors. more...