Evaluation and comparison of the dermatology program for medical students at the University of Chile with other national and foreign universities.

BACKGROUND: The National Examination of Knowledge in Medicine establishes the knowledge profile (PdC) a physician must possess to practice public medicine in Chile. However, no study has evaluated the perception of dermatology training regarding the acquisition of the minimum competencies required. This study described and compared the impressions of the dermatology training received by the University of Chile (UCh) graduates with graduates from other national and international faculties of medicine. MATERIALS AND METHODS: This was a cross-sectional study, based on a single survey model, applied via E-mail to registered physicians in an online database, with emphasis on UCh medicine graduates, from the generations 2012 to 2016. The data were collected anonymously, tabulated, and analyzed in MINITAB. RESULTS: From 908 UCh graduates, 141 surveys were answered (15.5%). Nine of 10 physicians considered "important" to obtain knowledge in dermatology. About 68.8% found the information they received was adequate. When comparing UCh graduates with other Chilean universities, UCh graduates had a slightly better impression of their training. When comparing Chilean versus foreign graduates, the latter presented a better perception of their preparation in cutaneous pathology. CONCLUSION: UCh graduates were satisfied with their dermatological training at the undergraduate level and felt better prepared than colleagues from other Chilean universities when facing cutaneous pathologies.

Analytical evaluation of an immunomagnetic separation PCR assay to detect pathogenic Leptospira in cattle urine samples obtained under field conditions.

Clinical manifestations of leptospirosis are diverse and very similar to other febrile diseases, hence early and accurate detection of subclinical infections is a key element in disease control. We evaluated immunomagnetic separation (IMS) capture technology coupled with a standard quantitative PCR (qPCR) system for the detection of pathogenic Leptospira in urine samples from 803 cows from dairy herds with a history of clinical cases of leptospirosis. The urine samples were first processed in a purification step, then subdivided into 2 subsamples, one that continued to DNA extraction and direct qPCR, and one that was pretreated by IMS before continuing to DNA extraction and qPCR. Overall, 133 of 803 (16.6%) samples were IMS-qPCR positive, whereas only 92 of 803 (11.5%) were positive when using direct qPCR. Statistically significant differences were observed between the mean estimated Leptospira load between the IMS-qPCR and the direct qPCR positive urine samples. The IMS-qPCR technology revealed a larger number of positive results and higher bacterial loads than direct qPCR. This difference is most likely the result of the high antigen-binding capacity and capture efficiency of the IMS system. The use of polyclonal antibodies produced by the inoculation of 3 synthetic peptides, which make up the extracellular regions of the LipL32 protein, provided a high detection capacity to the IMS-qPCR technique, resulting in performance superior to direct qPCR.

Telemedicina: acercando la reumatología a la población de la zona sur / Telemedicine: bringing rheumatology to the population of the chilean south zone

La Telemedicina constituye una herramienta que permite proporcionar atención médica especializada usando la tecnología de las telecomunicaciones.Entre mayo del 2015 y julio del 2017 se realizaron 1020 atenciones a través de esta modalidad, entre el Hospital Puerto Montt (HPM) y distintos centros de Atención primaria del SS Reloncaví.Se utilizaron dos modalidades de atención: asincrónica y sincrónica con presencia virtual del paciente.Se realizaron 1020 atenciones con una resolución inmediata en el 61,7% de los casos. Esta modalidad de atención implicó un ahorro de 139.412 Km, y por concepto de pasajes de $ 10.675.200 requeridos para el desplazamiento de los pacientes desde su lugar de origen al HPM.En lugares geográficamente distantes, la Telereumatología se convierte en una herramienta fundamental que permite expandir la cobertura de atenciones de salud por especialista, reducir las listas de espera, disminuir los tiempos de traslado y el costo que estos implican.

Telemedicine constitutes a tool that allows to provide specialized medical attention using telecommunications technology.Between May 2015 and July 2017, 1,020 care were carried out through this modality, between the Puerto Montt Hospital (HPM) and different primary care centers of the SS Reloncavi.Two care modalities were used: asynchronous and synchronous with the virtual presence of the patient.1020 visits were performed with immediate resolution in 61.7% of the cases.This care modality implied a saving of 139,412 km, and for the concept of passages of $ 10,675,200 required for the movement of patients from their place of origin to the HPM.In geographically remote places, Telerheumatology becomes a fundamental tool that allows expanding the coverage of health care by specialist, reducing waiting lists, reducing travel times and the cost that these imply.

The knocking down of the oncoprotein Golgi phosphoprotein 3 in T98G cells of glioblastoma multiforme disrupts cell migration by affecting focal adhesion dynamics in a focal adhesion kinase-dependent manner.

Golgi phosphoprotein 3 (GOLPH3) is a conserved protein of the Golgi apparatus that in humans has been implicated in tumorigenesis. However, the precise function of GOLPH3 in malignant transformation is still unknown. Nevertheless, clinicopathological data shows that in more than a dozen kinds of cancer, including gliomas, GOLPH3 could be found overexpressed, which correlates with poor prognosis. Experimental data shows that overexpression of GOLPH3 leads to transformation of primary cells and to tumor growth enhancement. Conversely, the knocking down of GOLPH3 in GOLPH3-overexpressing tumor cells reduces tumorigenic features, such as cell proliferation and cell migration and invasion. The cumulative evidence indicate that GOLPH3 is an oncoprotein that promotes tumorigenicity by a mechanism that impact at different levels in different types of cells, including the sorting of Golgi glycosyltransferases, signaling pathways, and the actin cytoskeleton. How GOLPH3 connects mechanistically these processes has not been determined yet. Further studies are important to have a more complete understanding of the role of GOLPH3 as oncoprotein. Given the genetic diversity in cancer, a still outstanding aspect is how in this inherent heterogeneity GOLPH3 could possibly exert its oncogenic function. We have aimed to evaluate the contribution of GOLPH3 overexpression in the malignant phenotype of different types of tumor cells. Here, we analyzed the effect on cell migration that resulted from stable, RNAi-mediated knocking down of GOLPH3 in T98G cells of glioblastoma multiforme, a human glioma cell line with unique features. We found that the reduction of GOLPH3 levels produced dramatic changes in cell morphology, involving rearrangements of the actin cytoskeleton and reduction in the number and dynamics of focal adhesions. These effects correlated with decreased cell migration and invasion due to affected persistence and directionality of cell motility. Moreover, the knocking down of GOLPH3 also caused a reduction in autoactivation of focal adhesion kinase (FAK), a cytoplasmic tyrosine kinase that regulates focal adhesions. Our data support a model in which GOLPH3 in T98G cells promotes cell migration by stimulating the activity of FAK.

Tamoxifen induces apoptosis and inhibits respiratory burst in equine neutrophils independently of estrogen receptors.

Neutrophils play an important role in the exacerbation and maintenance of severe equine asthma; persistent neutrophil activity and delayed apoptosis can be harmful to surrounding tissues. Tamoxifen (TX) is a nonsteroidal estrogen receptor modulator with immunomodulatory effects and induces early apoptosis of blood and bronchoalveolar lavage neutrophils from horses with acute lung inflammation. This study investigated if the in vitro effects of tamoxifen are produced by its action on nuclear (α and ß) and membrane (GPR30) estrogen receptors in healthy equine neutrophils. Results showed that TX inhibits neutrophil respiratory burst induced by opsonized zymosan in a dose-dependent manner. Nuclear (17-ß-Estradiol) and GPR30 cell membrane (G1) estrogen receptor agonists and their antagonists (ICI 182,780 and G15, respectively) do not block or reproduce the effect of TX. Therefore, TX does not inhibit respiratory burst through estrogen receptors. TX (8.5 µM) also increased phosphatidylserine translocation, a marker of early apoptosis, which did not occur with any of the estrogen receptor agonists or antagonists. Thus, tamoxifen generates dose-dependent inhibition of respiratory burst and increased early apoptosis in healthy equine neutrophils, independently of nuclear or membrane estrogen receptors. Further studies are necessary to explore the signaling pathways of tamoxifen-induced ROS inhibition and phosphatidylserine translocation.

Modulatory role of regulatory T cells in a murine model of severe equine asthma.

BACKGROUND: It is accepted that T regulatory cells (Treg) control different types of immune responses. In connection with this role, we have recently described an important increase in CD4+, CD25high, Foxp3+ lymphocytes in the airway system of horses coursing with an exacerbation of severe equine asthma (EA). To explore the potential role of this population in the resolution of EA inflammation, we used a murine experimental model in which airway neutrophilic inflammation, which is similar to that observed in EA, is induced in mice by continual exposure to Aspergillus fumigatus contaminated hay. This model has the advantage that in mice we may induce a reduction of the Treg population using low doses of cyclophosphamide (Cy). RESULTS: The results indicated that the percentage of Treg cells increased with allergen exposure, as in horses; and animals partially depleted of Treg cells by treatment with Cy showed increased airway inflammation, demonstrated by an increased percentage of neutrophils and specific immunoglobulins in bronchoalveolar lavage fluid (BALF). Furthermore, a histopathologic study of animals that were pretreated with Cy before antigenic challenge showed higher cellular infiltration in the lung and deeper remodeling changes in the bronchi, including epithelial and goblet cell hyperplasia as well as airway smooth muscle hypertrophy. CONCLUSION: In this murine model of EA, the reduced number and function of Treg induced by low doses of Cy, which directly correlates with increased airway inflammation and lung infiltration, indicates that Treg may play a major role in the regulation and resolution of EA.

Tamoxifen as a new therapeutic tool for neutrophilic lung inflammation.

BACKGROUND AND OBJECTIVE: Neutrophilic asthma is an important disease subgroup, including patients with severe phenotypes and erratic responses to standard treatments. Tamoxifen (TX), a selective estrogen receptor modulator (SERM) used as treatment of human breast cancer, has been shown to induce early apoptosis of equine blood and bronchoalveolar lavage fluid (BALF) neutrophils in vitro. Equine recurrent airway obstruction (RAO) is a naturally occurring neutrophilic condition, closely related with human asthma. Our purpose was to investigate the therapeutic potential of tamoxifen in horses with neutrophilic lung inflammation. METHODS: Twelve horses underwent acute lung inflammation through exposure to allergens known to cause RAO, after which they received treatment with either tamoxifen or dexamethasone. Outcome measures included evaluation of clinical signs, BALF cytology, and early apoptosis of blood and BALF neutrophils. RESULTS: Tamoxifen treatment decreased BALF neutrophil counts (65.3 ± 19.38% before treatment; 7.6 ± 4.5% 2 days post-treatment,; and 13.6 ± 9.3% 5 days post-treatment). A similar decrease was observed with dexamethasone treatment (48.6 ± 5.88% before treatment; 11.5 ± 8.1% 2 days post-treatment; 14.6 ± 10.3% 5 days post-treatment). Clinical and endoscopic scores improved in both treatment groups. Tamoxifen treatment significantly increased early apoptosis of peripheral blood neutrophils at 5 days post-treatment (27.04 ± 15.2%), and in BALF neutrophils at 2 and 5 days post-treatment (42.11 ± 11.67% and 48.98 ± 2.6%, respectively). CONCLUSION: Tamoxifen treatment in horses with induced acute pulmonary inflammation promoted early apoptosis of blood and BALF neutrophils, reduction in BALF neutrophils and improvement in the animals' clinical status.

Expression of the protein serum amyloid A in response to Aspergillus fumigatus in murine models of allergic airway inflammation.

BACKGROUND: Serum amyloid A (SAA) is an acute phase protein that is elevated in blood during inflammation. The role of this protein in allergic diseases of airways remains unclear. AIMS: The objective of this study was to evaluate the SAA in blood, lung and bronchial cells in a murine model of bronchial hypersensitivity to Aspergillus fumigatus. METHODS: To achieve this purpose, different groups of 5-month-old mice were housed in cages containing hay bedding that was contaminated with A. fumigatus and were kept in an isolation room for 16 days to allow for the induction of allergic airway inflammation. Subsequently, the mice were then exposed once again to Aspergillus spores at 0, 2, 8, 24 and 72 h, and they were bled to acquire serum and sacrificed to obtain bronchoalveolar lavage fluid (BALF) or lung tissues for analysis. SAA levels were measured in lung, serum and BALF by dot blot assay and RT-PCR (reverse transcription polymerase chain reaction). RESULTS: The results indicated that SAA protein levels increased in both serum and lung within 2-24h after mice were exposed to Aspergillus spores. Moreover, the SAA mRNA expression levels in the lungs and BALF cells demonstrated the same trend that was observed for the protein levels through the dot blot assay; in particular, SAA mRNA levels increased within the first hour after mice were exposed to A. fumigatus. CONCLUSIONS: In this allergic airway model, we conclude that A. fumigatus can induce an acute inflammatory response in the airways through the stimulation of the SAA protein, increasing its levels in serum, lung tissue and BALF samples during the early hours of exposure of mice that have been sensitised for this fungus.

Participation of T regulatory cells in equine recurrent airway obstruction.

Recurrent airway obstruction (RAO) is an equine immune-mediated disease with a high incidence worldwide. The aim of this work was to contribute to the understanding of RAO pathogenesis by studying T cells bearing regulatory markers in peripheral blood (PB) and in bronchoalveolar lavage fluid (BALF) recovered from the same group of susceptible horses before and after exposure to moldy hay, which has been shown to induce RAO signology in our horse herd. With this purpose, mononuclear cells were obtained from the BALF and PB from horses before and after antigenic challenge and were stained with fluorochrome-conjugated antibodies against CD4, CD25 and Foxp3 and subsequently analyzed by flow cytometry. The results indicated that the percentage of CD4+, Foxp3+ cells clearly increased in PB and BALF obtained from horses with RAO. In addition, the percentage of CD4+, CD25(high) cells was greatly augmented in BALF of RAO positive horses compared with a baseline. No changes were observed in the PB compartment. The percentage of CD4+, CD25(high), Foxp3+ cells in BALF increased in horses with active disease compared to horses in remission; this cell population also does not show changes in the PB compartment when RAO positive and RAO negative horses were compared. On the other hand, when the percentage of CD4, Foxp3 positive cells were compared with the percentage of CD4+, CD25(high) cells, the numbers were very similar. This observation was true for PB and BALF cells from non exposed horses as well as horses exposed to antigen. In all the experimental situations studied, the population expressing all of the markers CD4+, CD25(high), Foxp3+ represent only a minor percentage of CD4+, CD25(high) or CD4+, Foxp3 subpopulations; therefore, an significant number of CD4+, CD25(high), Foxp3- and CD4+, CD25(null), Foxp3+ cells must exist. Finally, we conclude that horses with RAO show an airway accumulation of T cells bearing regulatory markers that probably are modulating the course of this disease, and that these T cells may be involved in the resolution of immune-mediated bronchial inflammation.

Increased apoptosis of CD4 and CD8 T lymphocytes in the airways of horses with recurrent airway obstruction.

Recurrent airway obstruction (RAO, also known as equine heaves) is an inflammatory condition similar to human asthma caused by exposure of susceptible horses to poorly ventilated stable environments. The disease is characterized by neutrophilic airway inflammation, mucus hypersecretion and reversible bronchoconstriction. This inflammatory process is mediated by several factors, including antibodies, cytokines, resident cells of the airway and inflammatory cellular components that arrive in the respiratory tract. An increasing body of evidence has lent support to the concept that a dysregulation of T cell apoptosis may play a central role in the development of airway inflammation and the associated asthma. Therefore, the aim of this study was to investigate early and late apoptosis of CD4 and CD8 T cell subpopulations obtained from the airways of acute RAO-positive animals after exposure to hay/straw. The percentages of CD4 and CD8 T cells and their associated frequencies of apoptosis were quantified using flow cytometry. Hay/straw exposure induced clinical airway obstruction, airway neutrophilia and increased airway mucus production in RAO-positive horses. In addition, allergen exposure increased the percentage of CD4 T cells in RAO-positive horses as well as the frequency of early and late apoptosis in both CD4 and CD8 lymphocyte subpopulations. These results suggest that the higher frequency of lymphocyte apoptosis may play a role in disease progression of horses afflicted with RAO and may partially explain the characteristic remission of this pathological condition once the allergen source is removed. However, further studies are needed to clarify the role of T cell apoptosis in RAO-affected horses.

Detection of reaginic antibodies against Faenia rectivirgula from the serum of horses affected with Recurrent Airway Obstruction by an in vitro bioassay.

Reaginic antibodies, mainly of the IgE and some IgG subclasses, play an important role in the induction of type I immediate hypersensitivity reactions. These antibodies bind through their Fc fragment to high affinity receptors (FcεRI) present in the membrane of mast cells and basophils. Previously, several studies have investigated the role of reaginic antibodies in the pathogenesis of RAO. However, whereas immunological aspects of RAO have been extensively studied, the precise sequence of events is still not well understood and role of IgE in this disease still remains controversial. Therefore, in this study a bioassay was developed for reaginic antibody determination in serum from RAO-affected horses in order to determine the etiology of disease. The technique involves measuring in vitro calcium mobilization in RBL-2H3 cells following incubation with horse serum from RAO-affected or unaffected horses and one of the RAO antigens (Faenia rectivirgula). Results demonstrated that 15% of samples from the RAO-affected horses reacted positively in this in vitro bioassay, whereas the samples from unaffected horses did not. This bioassay indicates that reaginic antibodies could be involved in the immunological mechanism leading to RAO; and this technique may facilitate future research in other allergic diseases in horses.

Immunohistochemical evidences showing the presence of thymulin containing cells located in involuted thymus and in peripheral lymphoid organs.

Thymulin is a well-characterized thymic hormone that exists as a nonapeptide coupled to equimolar amounts of Zn2+. Thymulin is known to have multiple biological roles, including T cell differentiation, immune regulation, and analgesic functions. It has been shown that thymulin is produced by the reticulo-epithelial cells of the thymus, and it circulates in the blood from the moment of birth, maintain its serum level until puberty diminishing thereafter in life. To study the localization of this hormone, we prepared polyclonal and monoclonal antibodies against the commercial peptide and utilized immunocytochemical techniques for visualization. The results indicate that thymulin stains the thymic reticular cells, the outer layers of Hassall's corpuscles and a large round cellular type, which is keratin-negative and does not show affinity for the common leukocyte antigen (CD-45). In mice, this thymulin-positive cell remains in the thymus throughout life and even appears in relatively increased numbers in old involuted thymi. It also appears in thymus-dependent areas of the spleen and lymph nodes, demonstrating that at least one of the thymus cells containing this peptide can be found in peripheral lymphoid tissue.

In vitro bioassay to detect reaginic antibodies from the serum of horses affected with recurrent airway obstruction.

In horses, Recurrent Airway Obstruction (RAO) is an allergic disease that involves IgE mediated Type I Hypersensitivity responses. The development of this type of allergy involves a series of events that begins with reaginic antibodies, mainly IgE and some IgG subclasses. These reaginic antibodies bind with high affinity, via the Fc portion, to FcepsilonRI receptors on the membrane of mast cells and basophils. Once bound, environmental allergens cross-link the antibodies, which results in mast cell degranulation leading to the production of histamine and other chemical mediators that act together to induce airway inflammation. RAO-affected horses present with coughing, respiratory distress, airway obstruction and poor performance. The aspect of the RAO has been extensively studied, yet the precise sequence of events is still not well understood. Therefore, this study proposes a bioassay for reaginic antibody detection from horse serum of RAO-affected individuals, in order to determine the etiology of disease, which mediate immediate type reactions. The technique involves measuring in vitro calcium mobilization in RBL-2H3 cells following incubation with horse serum from affected or unaffected horses and one of the RAO antigens (Aspergillus fumigatus). The results presented here demonstrate that 30% of RAO-affected horses react positively in this in vitro bioassay, whereas unaffected horses do not. This bioassay may facilitate further research on RAO and other allergic diseases in horses.

Immunohistochemical evidences showing the presence of thymulin containing cells located in involuted thymus and in peripheral lymphoid organs

Thymulin is a well-characterized thymic hormone that exists as a nonapeptide coupled to equimolar amounts of Zn2+. Thymulin is known to have multiple biological roles, including T cell differentiation, immune regulation, and analgesic functions. It has been shown that thymulin is produced by the reticulo-epithelial cells of the thymus, and it circulates in the blood from the moment of birth, maintain its serum level until puberty diminishing thereafter in life. To study the localization of this hormone, we prepared polyclonal and monoclonal antibodies against the commercial peptide and utilized immunocytochemical techniques for visualization. The results indicate that thymulin stains the thymic reticular cells, the outer layers of Hassall's corpuscles and a large round cellular type, which is keratin-negative and does not show affinity for the common leukocyte antigen (CD-45). In mice, this thymulin-positive cell remains in the thymus throughout life and even appears in relatively increased numbers in old involuted thymi. It also appears in thymus-dependent areas of the spleen and lymph nodes, demonstrating that at least one of the thymus cells containing this peptide can be found in peripheral lymphoid tissue.

Cloning, expression and immunogenicity of the translation initiation factor 3 homologue of Brucella abortus.

The infC gene of Brucella abortus encoding the translation initiation factor 3 (IF3) was cloned, sequenced and expressed in Escherichia coli. The amino acid sequence analysis predicted a product with 74-80% identity with the IF3 proteins from Mesorhizobium loti, Sinorhizobium meliloti, Aurantimona sp. and Mesorhizobium sp. This protein also show 54% amino acid sequence identity with the E. coli IF3, sharing most of the residues which were described as responsible for the biological activity of this protein. Since we have previously reported the immunoprotective capacity of this Brucella protein, we stimulated lymphoid cells from animals immunized with purified recombinant Brucella IF3 protein "in vitro" with this antigen. The lymphocytes were able to mount a strong proliferative response with concomitant production of gamma interferon, but without the secretion of either IL-4 or antibodies. Thus, immunization with the Brucella recombinant IF3 protein promotes a TH-1 polarized response, allowing us to propose it as a promising candidate antigen for the development of subunit vaccines against Brucella.

Vaccination with live Escherichia coli expressing Brucella abortus Cu/Zn superoxide-dismutase: II. Induction of specific CD8+ cytotoxic lymphocytes and sensitized CD4+ IFN-gamma-producing cell.

Previously we reported that immunization with Escherichia coli DH5alpha-expressing Brucella abortus Cu/Zn superoxide dismutase [E. coli (pBSSOD)] induces a protective immune response in BALB/c mice. Here we studied the type of immune defense that the recombinant E. coli induces in mice using as our experimental model Brucella superoxide dismutase Cu/Zn presented by J744.A1 to sensitized lymphocytes as the target of specific lysis or as cytokine inductors. The results indicate that E. coli carrying the Cu/Zn gene was able to induce specific cytotoxic T cells, mainly from CD8(+) subpopulation and IFN-gamma-producing cells belonging in their vast majority to the CD4(+) subpopulation.

An RNA vaccine based on recombinant Semliki Forest virus particles expressing the Cu,Zn superoxide dismutase protein of Brucella abortus induces protective immunity in BALB/c mice.

We constructed infectious but replication-deficient Semliki Forest virus (SFV) particles carrying recombinant RNA encoding Brucella abortus Cu,Zn superoxide dismutase (SOD). The recombinant SFV particles (SFV-SOD particles) were then evaluated for their ability to induce a T-cell immune response and to protect BALB/c mice against a challenge with B. abortus 2308. Intraperitoneal injection of mice with recombinant SFV-SOD particles did not lead to the induction of SOD-specific antibodies, at least until week 6 after immunization (the end of the experiment). In vitro stimulation of splenocytes from the vaccinated mice with either recombinant Cu,Zn SOD (rSOD) or crude Brucella protein resulted in a T-cell proliferative response and the induction of gamma interferon secretion but not interleukin-4. In addition, the splenocytes exhibited significant levels of cytotoxic T-lymphocyte activity against Brucella-infected cells. The SFV-SOD particles, but not the control virus particles, induced a significant level of protection in BALB/c mice against challenge with B. abortus virulent strain 2308. These findings indicated that an SFV-based vector carrying the SOD gene has potential for use as a vaccine to induce resistance against B. abortus infections.

Determination of specific receptor sites for platelet activating factor in bovine neutrophils.

OBJECTIVE: To identify and characterize a platelet activating factor (PAF) receptor in bovine neutrophils by use of radioligand binding, reverse transcription-polymerase chain reaction (RT-PCR) assay, and western blot analysis. ANIMALS: 4 healthy adult cows. PROCEDURE: Bovine neutrophil membranes were isolated for association, dissociation, and saturation binding experiments with PAF labeled with hydrogen 3 (3H-PAF). The RT-PCR assay was performed with appropriate human primers, and western blot analysis was developed with a polyclonal antibody obtained from a peptide of bovine PAF receptor. RESULTS: Analysis of kinetic binding data supported a single class of PAF receptor. Binding of 3H-PAF to membrane preparations was selectively displaced by PAF and a nonhydrolyzable analogue of guanine triphosphate (Gpp[NH]p) and by lyso-PAF (a biologically inactive analogue of PAF) to a lesser extent. Among other PAF receptor antagonists, 14-deoxyandrographolide and WEB 2086 were the most effective in inhibiting 3H-PAF binding sites in neutrophil membranes; 2 lignans, schisandrin-A and gamma-schisandrin were also effective, but 2 gingkolides (BN52020 and BN52021) only mildly inhibited 3H-PAF binding. Results of RT-PCR assay and western blot analysis of neutrophil crude membranes confirmed the presence of a PAF receptor. CONCLUSIONS AND CLINICAL RELEVANCE: Results indicated that bovine neutrophils express only 1 type of PAF receptor, and it is likely that this receptor is involved in inflammatory responses. The most effective PAF antagonists were 14-deoxyandrographolide and WEB 2086; these PAF antagonists may be potentially useful in the treatment of inflammatory processes in cattle.

Intraspleen delivery of a DNA vaccine coding for superoxide dismutase (SOD) of Brucella abortus induces SOD-specific CD4+ and CD8+ T cells.

In the development of vaccines capable of providing immunity against brucellosis, Cu-Zn superoxide dismutase (SOD) has been demonstrated to be one of the protective immunogens of Brucella abortus. In an earlier study, we provided strong evidence that intramuscular injection with a plasmid DNA carrying the SOD gene (pcDNA-SOD) was able to induce a protective immune response. The present study was designed to characterize T-cell immune responses after an intraspleen (i.s.) vaccination of BALB/c mice with pcDNA-SOD. Animals vaccinated with pcDNA-SOD did not develop SOD-specific antibodies, at least until week 4 after immunization (the end of the experiment), and in vitro stimulation of their splenocytes with either recombinant Cu-Zn SOD or crude Brucella protein induced the secretion of gamma interferon (IFN-gamma), but not interleukin-4, and elicited the induction of cytotoxic-T-lymphocyte activity. Upon analyzing the SOD-specific T-cell responses, the pcDNA-SOD vaccination was found to be stimulating both CD4(+)- and CD8(+)-T-cell populations. However, only the CD4(+) population was able to produce IFN-gamma and only the CD8(+) population was able to induce cytotoxic activity. Nevertheless, although i.s. route vaccination induces a significant level of protection in BALB/c mice against challenge with the virulent B. abortus strain 2308, vaccination by the intramuscular route with a similar amount of plasmid DNA does not protect. Based on these results, we conclude that i.s. immunization with pcDNA-SOD vaccine efficiently induced a Th1 type of immune response and a protective response that could be related to IFN-gamma production and cytotoxic activity against infected cells by SOD-specific CD4(+) and CD8(+) T cells, respectively.