Type II interferon gamma (IFNγ) is a pleiotropic cytokine capable of modulating the innate and ad... more Type II interferon gamma (IFNγ) is a pleiotropic cytokine capable of modulating the innate and adaptive immune responses which has been widely characterized in several teleost families. In fish, IFNγ stimulates the expression of cytokines and chemokines associated with the pro-inflammatory response and enhances the production of nitrogen and oxygen reactive species in phagocytic cells. This work studied the effect of IFNγ on the expression of cell-surface markers on splenocytes of Atlantic salmon (Salmo salar). In vitro results showed that subpopulations of mononuclear splenocytes cultured for 15 days were capable of increasing gene expression and protein availability of cell-surface markers such as CD80/86, CD83 and MHC II, after being stimulated with recombinant IFNγ. These results were observed for subpopulations with characteristics associated with monocytes (51%), and features that could be related to lymphocytes (46.3%). In addition, a decrease in the expression of zbtb46 was ...
The high demand for supplies during the COVID19-pandemic has generated several stock-out of mater... more The high demand for supplies during the COVID19-pandemic has generated several stock-out of material and essential reagents needed to meet the current high demand for diagnosis in the worldwide population. In this way, there is limited information regarding the performance of different virus transport medium (VTM) for nasopharyngeal swab sampling (NPS) aimed for SARS-CoV-2 detection. We compared the RT-qPCR amplification profile of four different commercial transport medium kits, including DNA/RNA Shield, NAT, VTM, and Phosphate-buffered saline (PBS) transport medium, for NPSs samples from Central Metropolitan Health Service, Santiago, Chile. The RT-qPCR showed a slight lower RNase P Cq value of the samples preserved and transported in DNA/RNA Shield compared to NAT medium. By contrast, a marked increase in the RNase P Cq value was registered in the samples transported with VTM compared to DNA/RNA Shield medium. For PBS-preserved NPS, the performance of two strategies were assessed ...
Ectotherms choose the best thermal conditions to mount a successful immune response, a phenomenon... more Ectotherms choose the best thermal conditions to mount a successful immune response, a phenomenon known as behavioral fever. The cumulative evidence suggests that behavioral fever impacts positively upon lymphocyte proliferation, inflammatory cytokine expression, and other immune functions. In this study, we have explored how thermal choice during infection impacts upon underpinning molecular processes and how temperature increase is coupled to the immune response. Our results show that behavioral fever results in a widespread, plastic imprint on gene regulation, and lymphocyte proliferation. We further explored the possible contribution of histone modification and identified global associations between temperature and histone changes that suggest epigenetic remodeling as a result of behavioral fever. Together, these results highlight the critical importance of thermal choice in mobile ectotherms, particularly in response to an infection, and demonstrate the key role of epigenetic m...
Boletin Latinoamericano y del Caribe de Plantas Medicinales y Aromaticas
The intracellular bacteria Piscirickettsia salmonis is the most prevalent pathogen in the Chilean... more The intracellular bacteria Piscirickettsia salmonis is the most prevalent pathogen in the Chilean salmon industry, responsible for 50% of losses in recent years. So far, there are no effective treatments to control infections by this pathogen due to the emergence of antibiotics resistance. Therefore, it is extremely important to conduct research to find successful antibacterial therapies. In this paper, we evaluated the in vitro bactericidal activity of flavonoids and aromatic geranyl derivatives isolated from the resinous exudate of species Heliotropium filifolium, H. sinuatum y H. huascoense. The results showed that the compounds Filifolinone, Naringenine and 3-O-methylgalangine cause different percentage of mortality of bacteria and therefore they are good candidates to continue its evaluation in vitro and in vivo.
Proceedings of the National Academy of Sciences, 1992
This study describes an analysis of the interaction of individual amino acid residues of the vesi... more This study describes an analysis of the interaction of individual amino acid residues of the vesicular stomatitis virus (VSV) nucleocapsid antigenic octapeptide (N52-59; Arg-Gly-Tyr-Val-Tyr-Gln-Gly-Leu) with the H-2Kb molecule and T-cell receptors (TCRs). Tyr-3, Tyr-5, and Leu-8 were the positions in the peptide found to be H-2Kb contact residues by analyzing single alanine-substituted peptides in a competition assay with a Kb-restricted antigenic nonapeptide of Sendai virus. Arg-1, Gly-2, Val-4, Gln-6, and Gly-7 of the peptide were identified as putative TCR contact residues by testing the peptide analogs for their capacity to sensitize targets for VSV-specific cytolytic T-lymphocyte clones. The octamer N52-59 was the optimal length of the peptide required for binding to Kb. This peptide length requirement and the finding of an irregular interspersing of major histocompatibility complex and TCR contact residues are most consistent with the conclusion that the peptide is in an exten...
This study aims to identify at the expression level the immune-related genes associated with IPN-... more This study aims to identify at the expression level the immune-related genes associated with IPN-susceptible and resistant phenotypes in Atlantic salmon full-sibling families. We have analyzed thirty full-sibling families infected by immersion with IPNV and then classified as resistant or susceptible using a multivariate survival analysis based on a gamma-Cox frailty model and the Kaplan-Meier mortality curves. In four families within each group head kidneys were pooled for real-time PCR and one-color salmon-specific oligonucleotide microarray (21K) analysis at day 1 and 5 post-infection. Transcripts involved in innate response (IL-6, IFN-α), antigen presentation (HSP-70, HSP-90, MHC-I), TH1 response (IL-12, IFN-γ, CRFB6), immunosuppression (IL-10, TGF-β1) and leukocyte activation and migration (CCL-19, CD18) showed a differential expression pattern between both phenotypes, except in IL-6. In susceptible families, except for IFN-γ, the expressions dropped to basal values at day 5 post-infection. In resistant families, unlike susceptible families, levels remained high or increased (except for IL-6) at day 5. Transcriptomic analysis showed that both families have a clear differential expression pattern, resulting in a marked down-regulation in immune related genes involved in innate response, complement system, antigen recognition and activation of immune response in IPN-resistant. Down-regulation of genes, mainly related to tissue differentiation and protein degradation metabolism, was also observed in resistant families. We have identified an immune-related gene patterns associated with susceptibility and resistance to IPNV infection of Atlantic salmon. This suggests that a limited immune response is associated with resistant fish phenotype to IPNV challenge while a highly inflammatory but short response is associated with susceptibility.
Recent demonstrations of no changes in hypothalamic gonadotropin releasing hormone (GnRH) gene ex... more Recent demonstrations of no changes in hypothalamic gonadotropin releasing hormone (GnRH) gene expression and GnRH levels detected at the pituitary gland in diestrous and lactating rats, indicate that lactational hypogonadotropism in this species is not associated with inhibition of hypothalamic GnRH synthesis and secretion. Hypothalamic galanin potentiates GnRH effects on luteinizing hormone (LH) secretion in male and cycling rats. To explore the interaction between GnRH and galanin during lactation, we studied in vitro the effects of pulsatile stimulation with those peptides upon LH synthesis and secretion from rat pituitaries on diestrous 1 or day 10 of lactation. Hemipituitaries were separately incubated in 1 ml Dulbecco's Minimal Essential Medium supplemented with 1% penicillin-streptomycin and fetal calf serum, at 37 degrees C in 5% CO2-air. The hemipituitaries were stimulated during 12 h with hourly pulses, 6 min each, of (a) gonadotropin releasing hormone (GnRH 25 ng/pul...
Ribavirin is a synthetic nucleotide analog capable of inhibiting or even preventing some viral in... more Ribavirin is a synthetic nucleotide analog capable of inhibiting or even preventing some viral infections in mammals and also in fish. It has been seen by others that ribavirin by itself is able to stimulate the immune system of mammals, causing a differentiation of Tcells to T helper 1 cells (Th)-1. In this work, we evaluated the immune effect of ribavirin in vitro on kidney cells from Atlantic salmon and in vivo by oral administration of ribavirin to Atlantic salmon. For this purpose, the transcripts of immune molecules Tbet, GATA3, CD8, CD4, IFN␣, IFN␥, IL-4/13, IL-10, IL-12, IL-15 and TGF-B were quantified. The results show that ribavirin administered orally in food to Atlantic salmon increased IFN␥ and CD4 transcripts in the in vivo assays and, in addition, increased IL-12, IL-15 and CD8 in the in vitro analyses, indicating that the treatment stimulates a Th1 type response in salmon.
In the last years, the aquaculture crops have experienced an explosive and intensive growth, beca... more In the last years, the aquaculture crops have experienced an explosive and intensive growth, because of the high demand for protein. This growth has increased fish susceptibility to diseases and subsequent death. The constant biotic and abiotic changes experienced by fish species in culture are challenges that induce physiological, endocrine and immunological responses. These changes mitigate stress effects at the cellular level to maintain homeostasis. The effects of stress on the immune system have been studied for many years. While acute stress can have beneficial effects, chronic stress inhibits the immune response in mammals and teleost fish. In response to stress, a signaling cascade is triggered by the activation of neural circuits in the central nervous system because the hypothalamus is the central modulator of stress. This leads to the production of catecholamines, corticosteroid-releasing hormone, adrenocorticotropic hormone and glucocorticoids, which are the essential neuroendocrine mediators for this activation. Because stress situations are energetically demanding, the neuroendocrine signals are involved in metabolic support and will suppress the "less important" immune function. Understanding the cellular mechanisms of the neuroendocrine regulation of immunity in fish will allow the development of new pharmaceutical strategies and therapeutics for the prevention and treatment of diseases triggered by stress at all stages of fish cultures for commercial production.
Laminaran, fucoidan and sulfated galactan were extracted from the seaweeds Desmarestia distans, L... more Laminaran, fucoidan and sulfated galactan were extracted from the seaweeds Desmarestia distans, Lessonia vadosa, and Gigartina skottsbergii, respectively. Modified polysaccharides were prepared by desulfation, depolymerization, and sulfation. Antioxidant, anticoagulant and immunostimulating activities of native and modified polysaccharides were assayed in vitro. The oversulfated derivatives showed high antioxidant capacity towards oxygen radical assay; however, no direct relation between sulfate content and antioxidant capacity was found. Oversulfated polysaccharides presented higher antioxidant capacity towards hydroxyl radicals than the native polysaccharide. Regarding the ABTS þ radical cation assay moderate inhibition values (35.1-3.4%) were observed. The anticoagulant activity of native and modified polysaccharides was measured using the activated partial thromboplastin time assay; the native sulfated galactan from G. skottsbergii presented the highest value, close to that shown by heparin at similar concentrations. The immunostimulating activity of polysaccharides was measured through their effects on bone marrow-derived mice dendritic cell maturation. The native sulfated galactan from G. skottsbergii presented good dose-dependent activity inducing increased levels of MHC class II in dendritic cells.
Infectious pancreatic necrosis virus (IPNV) is a worldwide problem affecting both freshwater and ... more Infectious pancreatic necrosis virus (IPNV) is a worldwide problem affecting both freshwater and seawater fish. Vaccines developed against IPNV are not as efficient in the field as they are in tests. Moreover, research in the development of vaccines against IPNV has often shown that vaccines can stimulate the immune response of fish antibodies but do not protect efficiently against IPNV. In fact, sometimes dead infected fish show high antibody titers against IPNV. This suggests that the magnitude of total antibodies stimulated by the vaccine is not necessarily related to the level of protection against IPN, suggesting that a new method is needed to evaluate vaccine stimulation of the immune system. We propose in vitro evaluation of the non-specific cytotoxic cells (NCC) of the innate immune response, in addition to humoral specific response. Moreover, it is necessary to develop innovative methods to improve fish vaccines. In this work, IPNV replicative intermediaries (provirus) were used to inject rainbow trout fry, which is the most vulnerable state to IPNV. To evaluate the immune response triggered by this vaccine, NCC and total and neutralizing antibodies against IPNV and the provirus were determined. Results indicated that NCC activity in rainbow trout fry is triggered by IPNV infection. Both IPNV and the provirus stimulate humoral and NCC immune response in rainbow trout fry. Although the total antibodies triggered by the provirus were half of that triggered by IPNV infection, the number of neutralizing antibodies was similar in the two treatments. This suggests that the ratio of neutralizing antibodies is higher among the antibodies stimulated by provirons than among those stimulated by IPNV infection. Thus, immature provirus is sufficient to activate immune response and is a good candidate as an attenuated vaccine in rainbow trout fry. In addition, neutralizing antibodies, together with non-specific cytotoxic activity, are a more suitable strategy to evaluate new vaccines than humoral immune response alone.
Yeast pyruvate kinase was purified to near homogeneity and subjected to chemical modification by ... more Yeast pyruvate kinase was purified to near homogeneity and subjected to chemical modification by trinitrobenzenesulfonate and by pt P:-bis (5' pyridoxal) diphosphate. 2. Labeled peptides were isolated and their amino acid composition was determined. 3. The results suggest that yeast pyruvate kinase has an essential lysine residue, and that this residue is in a location equivalent to an essential lysine described in the muscle enzyme. 4. Protection experiments indicate that this lysine is located at the nucleotide binding site.
Proceedings of the National Academy of Sciences, 1992
To study the structure of a homogenous major histocompatibility complex (MHC) class I molecule co... more To study the structure of a homogenous major histocompatibility complex (MHC) class I molecule containing a single bound peptide, a complex of recombinant mouse H-2Kb, beta 2-microglobulin (beta 2m), and a fragment of the vesicular stomatitis virus (VSV) nuclear capsid protein, VSV-(N52-59) octapeptide (Arg-Gly-Tyr-Val-Tyr-Gln-Gly-Leu), was prepared by exploiting a high-yield bacterial expression system and in vitro cocomplex formation. The structure of mouse H-2Kb revealed its similarity to three human class I HLA molecules, consistent with the high primary sequence homology and common function of these peptide-presenting molecules. Electron density was located in the peptide-binding groove, to which a single peptide in a unique conformation was unambiguously fit. The peptide extends the length of the groove, parallel to the alpha-helices, and assumes an extended, mostly beta-strand conformation. The peptide is constrained within the groove by hydrogen bonding of its main-chain ato...
Pathogenic microorganisms, such as Neisseria gonorrhoeae, have developed mechanisms to alter epit... more Pathogenic microorganisms, such as Neisseria gonorrhoeae, have developed mechanisms to alter epithelial barriers in order to reach subepithelial tissues for host colonization. The aim of this study was to examine the effects of gonococci on cell junction complexes of genital epithelial cells of women. Polarized Ishikawa cells, a cell line derived from endometrial epithelium, were used for experimental infection. Infected cells displayed a spindle-like shape with an irregular distribution, indicating potential alteration of cell-cell contacts. Accordingly, analysis by confocal microscopy and cellular fractionation revealed that gonococci induced redistribution of the adherens junction proteins E-cadherin and its adapter protein β-catenin from the membrane to a cytoplasmic pool, with no significant differences in protein levels. In contrast, gonococcal infection did not induce modification of either expression or distribution of the tight junction proteins Occludin and ZO-1. Similar results were observed for Fallopian tube epithelia. Interestingly, infected Ishikawa cells also showed an altered pattern of actin cytoskeleton, observed in the form of stress fibers across the cytoplasm, which in turn matched a strong alteration on the expression of fibronectin, an adhesive glycoprotein component of extracellular matrix. Interestingly, using western blotting, activation of the ERK pathway was detected after gonococcal infection while p38 pathway was not activated. All effects were pili and Opa independent. Altogether, results indicated that gonococcus, as a mechanism of pathogenesis, induced disruption of junction complexes with early detaching of E-cadherin and β-catenin from the adherens junction complex, followed by a redistribution and reorganization of actin cytoskeleton and fibronectin within the extracellular matrix.
The role of FasL in the reproductive tissues of the non-pregnant mouse may be the induction of ap... more The role of FasL in the reproductive tissues of the non-pregnant mouse may be the induction of apoptosis of activated T cells for the prevention of unwanted inflammatory responses secondary to infection. This study was undertaken to identify cell and tissue types that typically express FasL in the uterus and oviduct of the mouse and to establish whether FasL on the surface of these cells was able to induce T cell apoptosis. FasL in the mouse uterus and oviduct was demonstrated using three independent methods: RT-PCR, Western blot and immunocytochemistry. The protein was present in the epithelial and mesenchymal cells of the uterus and showed a granular pattern in the apical epithelial portion. Although this suggests the presence of vesicles, surface expression was also detected by flow cytometry of isolated uterine cells. Exogenous administration of estradiol and progesterone had no significant effect on the expression and localization of FasL. The ability of uterine cells to induce FasL-dependent apoptosis of activated CD4+T cells was examined by incubation of phytohemagglutinin-treated T cells with cultured uterine cells. TUNEL and flow cytometric analyses showed that CD4+T cells experienced apoptosis after 5h of co-incubation. Neutralizing antibodies inhibited apoptosis demonstrating that a biologically active FasL is present in the reproductive tissues of the mouse. Results indicate that FasL is a biologically active molecule present in epithelial and mesenchymal cells of the uterus and the oviduct of the non-pregnant mouse that might restrain local immune response by induction of apoptosis of CD4+T lymphocytes.
Infection of the Fallopian tubes (FT) by Neisseria gonorrhoeae can lead to acute salpingitis, an ... more Infection of the Fallopian tubes (FT) by Neisseria gonorrhoeae can lead to acute salpingitis, an inflammatory condition, which is a major cause of infertility. Challenge of explants of human FT with gonococci induced mRNA expression and protein secretion for the proinflammatory cytokines interleukin (IL)-1α, IL-1β, and tumor necrosis factor alpha (TNF-α) but not for granulocyte-macrophage colony-stimulating factor. In contrast, FT expression of IL-6 and of the cytokine receptors IL-6R, TNF receptor I (TNF-RI), and TNF-RII was constitutive and was not increased by gonococcal challenge. These studies suggest that several proinflammatory cytokines are likely to contribute to the cell and tissue damage observed in gonococcal salpingitis.
Neisseria gonorrhoeae is a gram-negative diplococcus that in human beings produces gonorrhea. Muc... more Neisseria gonorrhoeae is a gram-negative diplococcus that in human beings produces gonorrhea. Much clinical evidence has led to the conclusion that gonococcus has important mechanisms to evade host immune functions; however, these mechanisms are only now beginning to be elucidated. In this study, we determined that the BALB/c mouse is a good animal model to study gonococcus infection and examined the immune response against the bacteria. We determined that after intravaginal inoculation of mice with Neisseria gonorrhoeae , the bacteria reached and invaded the upper female reproductive tissues and elicited a T-cell-specific immune response associated with a very weak humoral response, altogether resembling gonococcus infection and disease in women. Remarkably, in the draining lymph nodes of the genital tracts of infected mice, we found an increase of regulatory T lymphocytes, namely, transforming growth factor β1-positive CD4 + T cells and CD4 + CD25 + Foxp3 + T cells. Altogether, re...
Following infection with Neisseria gonorrhoeae , bacteria may ascend into the Fallopian tubes (FT... more Following infection with Neisseria gonorrhoeae , bacteria may ascend into the Fallopian tubes (FT) and induce salpingitis, a major cause of infertility. In the FT, interactions between mucosal epithelial cells and gonococci are pivotal events in the pathogen's infection cycle and the inflammatory response. In the current study, primary FT epithelial cells were infected in vitro with different multiplicities of infection (MOI) of Pil + Opa + gonococci. Bacteria showed a dose-dependent association with cells and induced the secretion of tumor necrosis factor alpha (TNF-α). A significant finding was that gonococcal infection (MOI = 1) induced apoptosis in approximately 30% of cells, whereas increasing numbers of bacteria (MOI = 10 to 100) did not induce apoptosis. Apoptosis was observed in only 11% of cells with associated bacteria, whereas >84% of cells with no adherent bacteria were apoptotic. TNF-α was a key contributor to apoptosis, since (i) culture supernatants from cells ...
Type II interferon gamma (IFNγ) is a pleiotropic cytokine capable of modulating the innate and ad... more Type II interferon gamma (IFNγ) is a pleiotropic cytokine capable of modulating the innate and adaptive immune responses which has been widely characterized in several teleost families. In fish, IFNγ stimulates the expression of cytokines and chemokines associated with the pro-inflammatory response and enhances the production of nitrogen and oxygen reactive species in phagocytic cells. This work studied the effect of IFNγ on the expression of cell-surface markers on splenocytes of Atlantic salmon (Salmo salar). In vitro results showed that subpopulations of mononuclear splenocytes cultured for 15 days were capable of increasing gene expression and protein availability of cell-surface markers such as CD80/86, CD83 and MHC II, after being stimulated with recombinant IFNγ. These results were observed for subpopulations with characteristics associated with monocytes (51%), and features that could be related to lymphocytes (46.3%). In addition, a decrease in the expression of zbtb46 was ...
The high demand for supplies during the COVID19-pandemic has generated several stock-out of mater... more The high demand for supplies during the COVID19-pandemic has generated several stock-out of material and essential reagents needed to meet the current high demand for diagnosis in the worldwide population. In this way, there is limited information regarding the performance of different virus transport medium (VTM) for nasopharyngeal swab sampling (NPS) aimed for SARS-CoV-2 detection. We compared the RT-qPCR amplification profile of four different commercial transport medium kits, including DNA/RNA Shield, NAT, VTM, and Phosphate-buffered saline (PBS) transport medium, for NPSs samples from Central Metropolitan Health Service, Santiago, Chile. The RT-qPCR showed a slight lower RNase P Cq value of the samples preserved and transported in DNA/RNA Shield compared to NAT medium. By contrast, a marked increase in the RNase P Cq value was registered in the samples transported with VTM compared to DNA/RNA Shield medium. For PBS-preserved NPS, the performance of two strategies were assessed ...
Ectotherms choose the best thermal conditions to mount a successful immune response, a phenomenon... more Ectotherms choose the best thermal conditions to mount a successful immune response, a phenomenon known as behavioral fever. The cumulative evidence suggests that behavioral fever impacts positively upon lymphocyte proliferation, inflammatory cytokine expression, and other immune functions. In this study, we have explored how thermal choice during infection impacts upon underpinning molecular processes and how temperature increase is coupled to the immune response. Our results show that behavioral fever results in a widespread, plastic imprint on gene regulation, and lymphocyte proliferation. We further explored the possible contribution of histone modification and identified global associations between temperature and histone changes that suggest epigenetic remodeling as a result of behavioral fever. Together, these results highlight the critical importance of thermal choice in mobile ectotherms, particularly in response to an infection, and demonstrate the key role of epigenetic m...
Boletin Latinoamericano y del Caribe de Plantas Medicinales y Aromaticas
The intracellular bacteria Piscirickettsia salmonis is the most prevalent pathogen in the Chilean... more The intracellular bacteria Piscirickettsia salmonis is the most prevalent pathogen in the Chilean salmon industry, responsible for 50% of losses in recent years. So far, there are no effective treatments to control infections by this pathogen due to the emergence of antibiotics resistance. Therefore, it is extremely important to conduct research to find successful antibacterial therapies. In this paper, we evaluated the in vitro bactericidal activity of flavonoids and aromatic geranyl derivatives isolated from the resinous exudate of species Heliotropium filifolium, H. sinuatum y H. huascoense. The results showed that the compounds Filifolinone, Naringenine and 3-O-methylgalangine cause different percentage of mortality of bacteria and therefore they are good candidates to continue its evaluation in vitro and in vivo.
Proceedings of the National Academy of Sciences, 1992
This study describes an analysis of the interaction of individual amino acid residues of the vesi... more This study describes an analysis of the interaction of individual amino acid residues of the vesicular stomatitis virus (VSV) nucleocapsid antigenic octapeptide (N52-59; Arg-Gly-Tyr-Val-Tyr-Gln-Gly-Leu) with the H-2Kb molecule and T-cell receptors (TCRs). Tyr-3, Tyr-5, and Leu-8 were the positions in the peptide found to be H-2Kb contact residues by analyzing single alanine-substituted peptides in a competition assay with a Kb-restricted antigenic nonapeptide of Sendai virus. Arg-1, Gly-2, Val-4, Gln-6, and Gly-7 of the peptide were identified as putative TCR contact residues by testing the peptide analogs for their capacity to sensitize targets for VSV-specific cytolytic T-lymphocyte clones. The octamer N52-59 was the optimal length of the peptide required for binding to Kb. This peptide length requirement and the finding of an irregular interspersing of major histocompatibility complex and TCR contact residues are most consistent with the conclusion that the peptide is in an exten...
This study aims to identify at the expression level the immune-related genes associated with IPN-... more This study aims to identify at the expression level the immune-related genes associated with IPN-susceptible and resistant phenotypes in Atlantic salmon full-sibling families. We have analyzed thirty full-sibling families infected by immersion with IPNV and then classified as resistant or susceptible using a multivariate survival analysis based on a gamma-Cox frailty model and the Kaplan-Meier mortality curves. In four families within each group head kidneys were pooled for real-time PCR and one-color salmon-specific oligonucleotide microarray (21K) analysis at day 1 and 5 post-infection. Transcripts involved in innate response (IL-6, IFN-α), antigen presentation (HSP-70, HSP-90, MHC-I), TH1 response (IL-12, IFN-γ, CRFB6), immunosuppression (IL-10, TGF-β1) and leukocyte activation and migration (CCL-19, CD18) showed a differential expression pattern between both phenotypes, except in IL-6. In susceptible families, except for IFN-γ, the expressions dropped to basal values at day 5 post-infection. In resistant families, unlike susceptible families, levels remained high or increased (except for IL-6) at day 5. Transcriptomic analysis showed that both families have a clear differential expression pattern, resulting in a marked down-regulation in immune related genes involved in innate response, complement system, antigen recognition and activation of immune response in IPN-resistant. Down-regulation of genes, mainly related to tissue differentiation and protein degradation metabolism, was also observed in resistant families. We have identified an immune-related gene patterns associated with susceptibility and resistance to IPNV infection of Atlantic salmon. This suggests that a limited immune response is associated with resistant fish phenotype to IPNV challenge while a highly inflammatory but short response is associated with susceptibility.
Recent demonstrations of no changes in hypothalamic gonadotropin releasing hormone (GnRH) gene ex... more Recent demonstrations of no changes in hypothalamic gonadotropin releasing hormone (GnRH) gene expression and GnRH levels detected at the pituitary gland in diestrous and lactating rats, indicate that lactational hypogonadotropism in this species is not associated with inhibition of hypothalamic GnRH synthesis and secretion. Hypothalamic galanin potentiates GnRH effects on luteinizing hormone (LH) secretion in male and cycling rats. To explore the interaction between GnRH and galanin during lactation, we studied in vitro the effects of pulsatile stimulation with those peptides upon LH synthesis and secretion from rat pituitaries on diestrous 1 or day 10 of lactation. Hemipituitaries were separately incubated in 1 ml Dulbecco's Minimal Essential Medium supplemented with 1% penicillin-streptomycin and fetal calf serum, at 37 degrees C in 5% CO2-air. The hemipituitaries were stimulated during 12 h with hourly pulses, 6 min each, of (a) gonadotropin releasing hormone (GnRH 25 ng/pul...
Ribavirin is a synthetic nucleotide analog capable of inhibiting or even preventing some viral in... more Ribavirin is a synthetic nucleotide analog capable of inhibiting or even preventing some viral infections in mammals and also in fish. It has been seen by others that ribavirin by itself is able to stimulate the immune system of mammals, causing a differentiation of Tcells to T helper 1 cells (Th)-1. In this work, we evaluated the immune effect of ribavirin in vitro on kidney cells from Atlantic salmon and in vivo by oral administration of ribavirin to Atlantic salmon. For this purpose, the transcripts of immune molecules Tbet, GATA3, CD8, CD4, IFN␣, IFN␥, IL-4/13, IL-10, IL-12, IL-15 and TGF-B were quantified. The results show that ribavirin administered orally in food to Atlantic salmon increased IFN␥ and CD4 transcripts in the in vivo assays and, in addition, increased IL-12, IL-15 and CD8 in the in vitro analyses, indicating that the treatment stimulates a Th1 type response in salmon.
In the last years, the aquaculture crops have experienced an explosive and intensive growth, beca... more In the last years, the aquaculture crops have experienced an explosive and intensive growth, because of the high demand for protein. This growth has increased fish susceptibility to diseases and subsequent death. The constant biotic and abiotic changes experienced by fish species in culture are challenges that induce physiological, endocrine and immunological responses. These changes mitigate stress effects at the cellular level to maintain homeostasis. The effects of stress on the immune system have been studied for many years. While acute stress can have beneficial effects, chronic stress inhibits the immune response in mammals and teleost fish. In response to stress, a signaling cascade is triggered by the activation of neural circuits in the central nervous system because the hypothalamus is the central modulator of stress. This leads to the production of catecholamines, corticosteroid-releasing hormone, adrenocorticotropic hormone and glucocorticoids, which are the essential neuroendocrine mediators for this activation. Because stress situations are energetically demanding, the neuroendocrine signals are involved in metabolic support and will suppress the "less important" immune function. Understanding the cellular mechanisms of the neuroendocrine regulation of immunity in fish will allow the development of new pharmaceutical strategies and therapeutics for the prevention and treatment of diseases triggered by stress at all stages of fish cultures for commercial production.
Laminaran, fucoidan and sulfated galactan were extracted from the seaweeds Desmarestia distans, L... more Laminaran, fucoidan and sulfated galactan were extracted from the seaweeds Desmarestia distans, Lessonia vadosa, and Gigartina skottsbergii, respectively. Modified polysaccharides were prepared by desulfation, depolymerization, and sulfation. Antioxidant, anticoagulant and immunostimulating activities of native and modified polysaccharides were assayed in vitro. The oversulfated derivatives showed high antioxidant capacity towards oxygen radical assay; however, no direct relation between sulfate content and antioxidant capacity was found. Oversulfated polysaccharides presented higher antioxidant capacity towards hydroxyl radicals than the native polysaccharide. Regarding the ABTS þ radical cation assay moderate inhibition values (35.1-3.4%) were observed. The anticoagulant activity of native and modified polysaccharides was measured using the activated partial thromboplastin time assay; the native sulfated galactan from G. skottsbergii presented the highest value, close to that shown by heparin at similar concentrations. The immunostimulating activity of polysaccharides was measured through their effects on bone marrow-derived mice dendritic cell maturation. The native sulfated galactan from G. skottsbergii presented good dose-dependent activity inducing increased levels of MHC class II in dendritic cells.
Infectious pancreatic necrosis virus (IPNV) is a worldwide problem affecting both freshwater and ... more Infectious pancreatic necrosis virus (IPNV) is a worldwide problem affecting both freshwater and seawater fish. Vaccines developed against IPNV are not as efficient in the field as they are in tests. Moreover, research in the development of vaccines against IPNV has often shown that vaccines can stimulate the immune response of fish antibodies but do not protect efficiently against IPNV. In fact, sometimes dead infected fish show high antibody titers against IPNV. This suggests that the magnitude of total antibodies stimulated by the vaccine is not necessarily related to the level of protection against IPN, suggesting that a new method is needed to evaluate vaccine stimulation of the immune system. We propose in vitro evaluation of the non-specific cytotoxic cells (NCC) of the innate immune response, in addition to humoral specific response. Moreover, it is necessary to develop innovative methods to improve fish vaccines. In this work, IPNV replicative intermediaries (provirus) were used to inject rainbow trout fry, which is the most vulnerable state to IPNV. To evaluate the immune response triggered by this vaccine, NCC and total and neutralizing antibodies against IPNV and the provirus were determined. Results indicated that NCC activity in rainbow trout fry is triggered by IPNV infection. Both IPNV and the provirus stimulate humoral and NCC immune response in rainbow trout fry. Although the total antibodies triggered by the provirus were half of that triggered by IPNV infection, the number of neutralizing antibodies was similar in the two treatments. This suggests that the ratio of neutralizing antibodies is higher among the antibodies stimulated by provirons than among those stimulated by IPNV infection. Thus, immature provirus is sufficient to activate immune response and is a good candidate as an attenuated vaccine in rainbow trout fry. In addition, neutralizing antibodies, together with non-specific cytotoxic activity, are a more suitable strategy to evaluate new vaccines than humoral immune response alone.
Yeast pyruvate kinase was purified to near homogeneity and subjected to chemical modification by ... more Yeast pyruvate kinase was purified to near homogeneity and subjected to chemical modification by trinitrobenzenesulfonate and by pt P:-bis (5' pyridoxal) diphosphate. 2. Labeled peptides were isolated and their amino acid composition was determined. 3. The results suggest that yeast pyruvate kinase has an essential lysine residue, and that this residue is in a location equivalent to an essential lysine described in the muscle enzyme. 4. Protection experiments indicate that this lysine is located at the nucleotide binding site.
Proceedings of the National Academy of Sciences, 1992
To study the structure of a homogenous major histocompatibility complex (MHC) class I molecule co... more To study the structure of a homogenous major histocompatibility complex (MHC) class I molecule containing a single bound peptide, a complex of recombinant mouse H-2Kb, beta 2-microglobulin (beta 2m), and a fragment of the vesicular stomatitis virus (VSV) nuclear capsid protein, VSV-(N52-59) octapeptide (Arg-Gly-Tyr-Val-Tyr-Gln-Gly-Leu), was prepared by exploiting a high-yield bacterial expression system and in vitro cocomplex formation. The structure of mouse H-2Kb revealed its similarity to three human class I HLA molecules, consistent with the high primary sequence homology and common function of these peptide-presenting molecules. Electron density was located in the peptide-binding groove, to which a single peptide in a unique conformation was unambiguously fit. The peptide extends the length of the groove, parallel to the alpha-helices, and assumes an extended, mostly beta-strand conformation. The peptide is constrained within the groove by hydrogen bonding of its main-chain ato...
Pathogenic microorganisms, such as Neisseria gonorrhoeae, have developed mechanisms to alter epit... more Pathogenic microorganisms, such as Neisseria gonorrhoeae, have developed mechanisms to alter epithelial barriers in order to reach subepithelial tissues for host colonization. The aim of this study was to examine the effects of gonococci on cell junction complexes of genital epithelial cells of women. Polarized Ishikawa cells, a cell line derived from endometrial epithelium, were used for experimental infection. Infected cells displayed a spindle-like shape with an irregular distribution, indicating potential alteration of cell-cell contacts. Accordingly, analysis by confocal microscopy and cellular fractionation revealed that gonococci induced redistribution of the adherens junction proteins E-cadherin and its adapter protein β-catenin from the membrane to a cytoplasmic pool, with no significant differences in protein levels. In contrast, gonococcal infection did not induce modification of either expression or distribution of the tight junction proteins Occludin and ZO-1. Similar results were observed for Fallopian tube epithelia. Interestingly, infected Ishikawa cells also showed an altered pattern of actin cytoskeleton, observed in the form of stress fibers across the cytoplasm, which in turn matched a strong alteration on the expression of fibronectin, an adhesive glycoprotein component of extracellular matrix. Interestingly, using western blotting, activation of the ERK pathway was detected after gonococcal infection while p38 pathway was not activated. All effects were pili and Opa independent. Altogether, results indicated that gonococcus, as a mechanism of pathogenesis, induced disruption of junction complexes with early detaching of E-cadherin and β-catenin from the adherens junction complex, followed by a redistribution and reorganization of actin cytoskeleton and fibronectin within the extracellular matrix.
The role of FasL in the reproductive tissues of the non-pregnant mouse may be the induction of ap... more The role of FasL in the reproductive tissues of the non-pregnant mouse may be the induction of apoptosis of activated T cells for the prevention of unwanted inflammatory responses secondary to infection. This study was undertaken to identify cell and tissue types that typically express FasL in the uterus and oviduct of the mouse and to establish whether FasL on the surface of these cells was able to induce T cell apoptosis. FasL in the mouse uterus and oviduct was demonstrated using three independent methods: RT-PCR, Western blot and immunocytochemistry. The protein was present in the epithelial and mesenchymal cells of the uterus and showed a granular pattern in the apical epithelial portion. Although this suggests the presence of vesicles, surface expression was also detected by flow cytometry of isolated uterine cells. Exogenous administration of estradiol and progesterone had no significant effect on the expression and localization of FasL. The ability of uterine cells to induce FasL-dependent apoptosis of activated CD4+T cells was examined by incubation of phytohemagglutinin-treated T cells with cultured uterine cells. TUNEL and flow cytometric analyses showed that CD4+T cells experienced apoptosis after 5h of co-incubation. Neutralizing antibodies inhibited apoptosis demonstrating that a biologically active FasL is present in the reproductive tissues of the mouse. Results indicate that FasL is a biologically active molecule present in epithelial and mesenchymal cells of the uterus and the oviduct of the non-pregnant mouse that might restrain local immune response by induction of apoptosis of CD4+T lymphocytes.
Infection of the Fallopian tubes (FT) by Neisseria gonorrhoeae can lead to acute salpingitis, an ... more Infection of the Fallopian tubes (FT) by Neisseria gonorrhoeae can lead to acute salpingitis, an inflammatory condition, which is a major cause of infertility. Challenge of explants of human FT with gonococci induced mRNA expression and protein secretion for the proinflammatory cytokines interleukin (IL)-1α, IL-1β, and tumor necrosis factor alpha (TNF-α) but not for granulocyte-macrophage colony-stimulating factor. In contrast, FT expression of IL-6 and of the cytokine receptors IL-6R, TNF receptor I (TNF-RI), and TNF-RII was constitutive and was not increased by gonococcal challenge. These studies suggest that several proinflammatory cytokines are likely to contribute to the cell and tissue damage observed in gonococcal salpingitis.
Neisseria gonorrhoeae is a gram-negative diplococcus that in human beings produces gonorrhea. Muc... more Neisseria gonorrhoeae is a gram-negative diplococcus that in human beings produces gonorrhea. Much clinical evidence has led to the conclusion that gonococcus has important mechanisms to evade host immune functions; however, these mechanisms are only now beginning to be elucidated. In this study, we determined that the BALB/c mouse is a good animal model to study gonococcus infection and examined the immune response against the bacteria. We determined that after intravaginal inoculation of mice with Neisseria gonorrhoeae , the bacteria reached and invaded the upper female reproductive tissues and elicited a T-cell-specific immune response associated with a very weak humoral response, altogether resembling gonococcus infection and disease in women. Remarkably, in the draining lymph nodes of the genital tracts of infected mice, we found an increase of regulatory T lymphocytes, namely, transforming growth factor β1-positive CD4 + T cells and CD4 + CD25 + Foxp3 + T cells. Altogether, re...
Following infection with Neisseria gonorrhoeae , bacteria may ascend into the Fallopian tubes (FT... more Following infection with Neisseria gonorrhoeae , bacteria may ascend into the Fallopian tubes (FT) and induce salpingitis, a major cause of infertility. In the FT, interactions between mucosal epithelial cells and gonococci are pivotal events in the pathogen's infection cycle and the inflammatory response. In the current study, primary FT epithelial cells were infected in vitro with different multiplicities of infection (MOI) of Pil + Opa + gonococci. Bacteria showed a dose-dependent association with cells and induced the secretion of tumor necrosis factor alpha (TNF-α). A significant finding was that gonococcal infection (MOI = 1) induced apoptosis in approximately 30% of cells, whereas increasing numbers of bacteria (MOI = 10 to 100) did not induce apoptosis. Apoptosis was observed in only 11% of cells with associated bacteria, whereas >84% of cells with no adherent bacteria were apoptotic. TNF-α was a key contributor to apoptosis, since (i) culture supernatants from cells ...
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Papers by Monica Imarai