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In this study, we examined the cytokine immune response against two proteins of infectious pancreatic necrosis virus (IPNV) in rainbow trout (Oncorhynchus mykiss), the virion-associated RNA polymerase VP1 and VP2-Flagellin (VP2-Flg) fusion protein. Since VP1 is not a structural protein, we hypothesize it can induce cellular immunity, an essential mechanism of the antiviral response. At the same time, the fusion construction VP2-Flg could be highly immunogenic due to the presence of the flagellin used as an adjuvant. Fish were immunized with the corresponding antigen in Montanide™, and the gene expression of a set of marker genes of Th1, Th2, and the immune regulatory response was quantified in the head kidney of immunized and control fish. Results indicate that VP1 induced upregulation of ifn-γ, il-12p40c, il-4/13a, il-4/13b2, il-10a, and tgf-β1 in immunized fish. Expression of il-2a did not change in treated fish at the times tested. The antigen-dependent response was analysed by in vitro restimulation of head kidney leukocytes. In this assay, the group of cytokines upregulated after VP1-restimulation was consistent with those upregulated in the head kidney in vivo. Interestingly, VP1 induced il-2a expression after in vitro restimulation. The analysis of sorted lymphocytes showed that the increase of cytokines occurred in CD4-1

International audience; The identification and tracking of SARS-CoV-2 infected patients in the general population are essential components of the global strategy to limit the COVID-19 viral spread, specifically for maintaining traceability and suppressing the resurgence of local outbreaks. Public health programs that include continuous RT-qPCR testing for COVID-19 in the general population, viral sequencing, and genomic surveillance for highly contagious forms of the virus have allowed for the identification of SARS-CoV-2 infections and reinfections. This work identified SARS-CoV-2 reinfection in a homeless person, which occurred 58 days after the first COVID-19 diagnosis. Genomic sequencing identified a different Nextstrain classification clade (20A and 20B) and PANGO lineage, with a divergence of 4 single nucleotide variants (SNVs) in S and ORF1ab genes, suggesting reinfection by different viral variants. This study is the first from the great metropolitan area of Santiago, Chile, one of the top ten countries in the world to live during the COVID-19 pandemic. We support the importance of performing intensive genomic surveillance programs in the whole population and high-risk groups, such as homeless people, nearly 20 thousand people in Chile, and have limited access to health care services and poor viral traceability.

In poikilothermic vertebrates, seasonality influences different immunological parameters such as leukocyte numbers, phagocytic activity, and antibody titers. This phenomenon has been described in different teleost species, with immunological parameters peaking during warmer months and decreased levels during winter. In this study, the cellular immune responses of rainbow trout

Simple Summary The thymus is the immune organ producing T lymphocytes that are essential to create immunity after encountering pathogens or vaccination. This review summarizes the thymus localization and histological studies, cell composition, and function in teleost fishes. We also describe how seasonal changes, photoperiod, water temperature fluctuations, and hormones can affect thymus development in fish species. Overall, the information helps identify future studies needed to understand thymus function in fish species and the immune system’s evolutionary origins. Since fish are exposed to pathogens, especially under aquaculture conditions, knowledge about the fish thymus and T lymphocyte can also help improve fish farming protocols, considering intrinsic and environmental conditions that can contribute to achieving the best vaccine responsiveness for disease resistance. Abstract The thymus in vertebrates plays a critical role in producing functionally competent T-lymphocytes. Phylogenetically, the thymus emerges early during evolution in jawed cartilaginous fish, and it is usually a bilateral organ placed subcutaneously at the dorsal commissure of the operculum. In this review, we summarize the current understanding of the thymus localization, histology studies, cell composition, and function in teleost fishes. Furthermore, we consider environmental factors that affect thymus development, such as seasonal changes, photoperiod, water temperature fluctuations and hormones. Further analysis of the thymus cell distribution and function will help us understand how key stages for developing functional T cells occur in fish, and how thymus dynamics can be modulated by external factors like photoperiod. Overall, the information presented here helps identify the knowledge gaps and future steps needed for a better understanding of the immunobiology of fish thymus.

Interleukin (IL)-4 and IL-13 play a central role in T helper 2 immune response in mammals. The cell signalling is mediated by the type I heterodimeric receptor containing the IL-4Rα and γC chains, and the type II receptors formed by IL-4Rα and IL-13Rα1. In salmonid species, three paralogues of the IL-4 and IL-13 cytokines have been reported, il-4/13a, il-4/13b1 and il-4/13b2. In regard to receptors, two paralogues of each IL-4/13 receptor chains have been identified in rainbow trout while five genes named γc1, il-4rα, il-13rα1a, il-13rα1b, and il-13rα2 have identified in Atlantic salmon. Since Atlantic salmon is an important farmed fish species, the aim of this work was to get new insights into distribution, structure and expression regulation of the IL-4/13 receptors in salmon. By using qRT-PCR, it was shown that all γc1, il-4rα, il-13rα1a, il-13rα1b, and il-13rα2 receptor chains were expressed in lymphoid and non-lymphoid tissues of healthy salmon, nonetheless γC expression was higher in lymphoid than non-lymphoid tissues. The in silico structural analysis and homology modelling of the predicted receptor proteins showed that domains and most motifs present in the superior vertebrate chains are conserved in salmon suggesting a conserved role for these receptor chains. Only IL-13Rα1B is a receptor chain with a unique structure that seem not to be present in higher vertebrates but in fish species. In order to determine the regulatory role of IL-4/13 on the expression of receptor chains, Atlantic salmon il-4/13A gene was synthetized and cloned in pET15b. The recombinant IL-4/13A was produced in E. coli and the activity of the purified cytokine was confirmed in vitro. The regulatory role of IL-4/13A on the expression of their potential receptors was tested in salmon receiving the recombinant cytokine and effects were compared with those of the control group. The results showed that IL-4/13A induced the expression of its own gene and GATA-3, in the head kidney of fish but not in the spleen, while IL-10 increased in both lymphoid organs indicating a regulatory role of this cytokine on the induction of Th2 responses in salmon. IFN-γ and MHC class II were also later induced in head kidney. In regard to the expression of the receptor chains, IL-4/13A upregulated the expression of γC, IL-13Rα1A and IL-13Rα2A in the spleen but not in the head kidney of salmon, indicating a role on the modulation of cell signalling for the Th2 response. Furthermore, Piscirickettsia salmonis infection of Atlantic salmon occurred with an increase of γC and IL-13Rα1A suggesting a potential role of the IL-4/13 system in bacterial immunity or pathogenesis. This study contributes to a better understanding of the IL-4/13A system in salmon, which as a key axis for Th2 response may be involved not only in pathogen elimination but also in adaptive immune repair that seems critical tolerance to infectious diseases.

In Chile, the salmon and trout farmed fishing industries have rapidly grown during the last years, becoming one of the most important economic sources for the country. However, infectious diseases caused by bacteria, virus, mycoses and parasites, result in losses of up to 700 million dollars per year for the Chilean aquaculture production with the consequent increase of antibiotic and antiparasitic usage. After 30 years of its first appearance, the main salmon health problem is still the salmonid rickettsial septicaemia (SRS), which together with other disease outbreaks, reveal that vaccines do not provide acceptable levels of long-lasting immune protection in the field. On the other hand, due to the large dependence of the industry on salmonids production, the Chilean government promoted the Aquaculture diversification program by 2009, which includes new species such as Merluccius australis, Cilus gilberti and Genypterus chilensis, however, specific research regarding the immune system and vaccine development are issues that still need to be addressed and must be considered as important as the farm production technologies for new fish species. Based on the experience acquired from the salmonid fish farming, should be mandatory an effort to study the immune system of the new species to develop knowledge for vaccination approaches, aiming to protect these aquaculture species before diseases outbreaks may occur. This review focuses on the current status of the Chilean aquaculture industry, the challenges related to emerging and re-emerging microbial pathogens on salmonid fish farming, and the resulting needs in the development of immune protection by rational designed vaccines. We also discussed about what we have learn from 25 years of salmonid researches and what can be applied to the new Chilean farmed species on immunology and vaccinology.

Rapid and high quality preparation of peripheral blood leucocytes (PBL) is important in fish immunology research and in particular for fish vaccine development, where multiple immune parameters can be monitored on the same fish over time. Fish PBL are currently prepared by density separation using Percoll or Hispaque-1.077, which is time consuming, costly and prone to erythrocyte contamination. We present here a modified PBL preparation method that includes a 20 s hypotonic lysis of erythrocytes and a subsequent separation of PBL from cell debris by a cell strainer. This method is simple, rapid and cost effective. The PBL obtained are similar in cellular composition to those prepared by density separation but have less erythrocyte contamination as demonstrated by FACS analysis and the expression of cell marker genes. Marker gene analysis also suggested that PBL prepared by hypotonic lysis are superior to those obtained by the gradient method in that some high-density cells (certain B cell types and neutrophils) might be lost using the latter. The PBL prepared in this way can proliferate in response to the T cell mitogen PHA, and both lymphoid and myeloid cells can phagocytose fluorescent beads and bacteria, with the latter enhanced by treatment with pro-inflammatory cytokines (IL-1β and IL-6). Furthermore, the PBL can respond to stimulation with PAMPs (LPS, poly I:C) and cytokines (IL-1β and IFNγ) in terms of upregulation of proinflammatory cytokine gene expression. Such data demonstrate the utility of this approach (hypotonic lysis of erythrocytes) for PBL isolation and will enable more studies of their role in disease protection in future immunological and vaccine development research in fish.

In superior vertebrates, Interleukin 4 (IL-4) and Interleukin 13 (IL-13) play key and diverse roles to support immune responses acting on cell surface receptors. When stimulated, receptors activate intracellular signalling cascades switching cell phenotypes according to stimuli. In teleost fish, Interleukin 4/13 (IL-4/13) is the ancestral family cytokine related to both IL-4 and IL-13. Every private and common receptor subunit for IL-4/13 have in fish at least two paralogues and, as in mammals, soluble forms are also part of the receptor system. Reports for findings of fish IL-4/13 receptors have covered comparative analysis, transcriptomic profiles and to a lesser extent, functional analysis regarding ligand-receptor interactions and their biological effects. This review addresses available information from fish IL-4/13 receptors and discusses overall implications on teleost immunity, summarized gene induction strategies and pathogen-induced gene modulation, which may be useful tools to enhance immune response. Additionally, we present novel coding sequences for Atlantic salmon (Salmo salar) common gamma chain receptor (γC), Interleukin 13 receptor alpha 1A chain (IL-13Rα1A) and Interleukin 13 receptor alpha 1B chain (IL-13Rα1B).

Synergy could be an effective strategy to potentiate and recover antibiotics nowadays useless in clinical treatments against multi-resistant bacteria. In this study, synergic interactions between antibiotics and grape pomace extract that contains high concentration of phenolic compounds were evaluated by the checkerboard method in clinical isolates of Staphylococcus aureus and Escherichia coli. To define which component of the extract is responsible for the synergic effect, phenolic compounds were identified by RP-HPLC and their relative abundance was determined. Combinations of extract with pure compounds identified there in were also evaluated. Results showed that the grape pomace extract combined with representatives of different classes of antibiotics as β-lactam, quinolone, fluoroquinolone, tetracycline and amphenicol act in synergy in all S. aureus and E. coli strains tested with FICI values varying from 0.031 to 0.155. The minimal inhibitory concentration (MIC) was reduced 4 to 75 times. The most abundant phenolic compounds identified in the extract were quercetin, gallic acid, protocatechuic acid and luteolin with relative abundance of 26.3, 24.4, 16.7 and 11.4%, respectively. All combinations of the extract with the components also showed synergy with FICI values varying from 0.031 to 0.5 and MIC reductions of 4 to 125 times with both bacteria strains. The relative abundance of phenolic compounds has no correlation with the obtained synergic effect, suggesting that the mechanism by which the synergic effect occurs is by a multi-objective action. It was also shown that combinations of grape pomace extract with antibiotics are not toxic for the HeLa cell line at concentrations in which the synergistic effect was observed (47 μg/mL of extract and 0.6-375 μg/mL antibiotics). Therefore, these combinations are good candidates for testing in animal models in order to enhance the effect of antibiotics of different classes and thus restore the currently unused clinical antibiotics due to the phenomenon of resistance. Moreover, the use of grape pomace is a good and low-cost alternative for this purpose being a waste residue of the wine industry.

Neisseria gonorrhoeaeis the etiological agent of gonorrhoea, which is a sexually transmitted disease widespread throughout the world.N. gonorrhoeaedoes not improve immune response in patients with reinfection, suggesting that gonococcus displays several mechanisms to evade immune response and survive in the host.N. gonorrhoeaeis able to suppress the protective immune response at different levels, such as B and T lymphocytes and dendritic cells. In this study, we determined whetherN. gonorrhoeaedirectly conditions the phenotype of RAW 264.7 murine macrophage cell line and its response. We established that gonococcus was effectively phagocytosed by the RAW 264.7 cells and upregulates production of immunoregulatory cytokines (IL-10 and TGF-β1) but not the production of proinflammatory cytokine TNF-α, indicating that gonococcus induces a shift towards anti-inflammatory cytokine production. Moreover,N. gonorrhoeaedid not induce significant upregulation of costimulatory CD86 and MHC class II molecules. We also showed thatN. gonorrhoeaeinfected macrophage cell line fails to elicit proliferative CD4+ response. This implies that macrophage that can phagocytose gonococcus do not display proper antigen-presenting functions. These results indicate thatN. gonorrhoeaeinduces a tolerogenic phenotype in antigen-presenting cells, which seems to be one of the mechanisms to induce evasion of immune response.

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.

IPNV is the agent of a well-characterized acute disease that produces a systemic infection and high mortality in farmed fish species and persistent infection in surviving fish after outbreaks. Because modulation of the host expression of pro and anti-inflammatory cytokines can help establish persistence, in this study, we examined the expression of IL-1β, IL-8, IFNα1 and IL-10 during acute and persistent IPNV infection of Atlantic salmon. Results showed that IPNV infection induces an increase of the IFNα1 and IL-10 mRNA levels in the spleen and head kidney (HK) of fish after acute experimental infection. Levels of the pro-inflammatory cytokines IL-1β and IL-8 did not rise in the spleen although an increase of IL-1β, but not of IL-8, was observed in head kidney. In carrier asymptomatic salmon, cytokine gene expression of IFNα1 in the spleen and IL-10 in head kidney were also significantly higher than expression in non-carrier fish. Interestingly, a decrease of IL-8 expression was also observed. IPNV infection of SHK-1, which is a macrophage-like cell line of salmon, also induced an increase of expression of the anti-inflammatory cytokine IL-10 with no effects on the expression of IL-1β and IL-8. The effects are induced by an unknown mechanism during viral infection because poly I:C and the viral genomic dsRNA showed the opposite effects on cytokine expression in SHK-1 cells. In summary, IPNV always induces up-regulation of the anti-inflammatory cytokine IL-10 in Atlantic salmon. As this is accompanied by a lack of induction of the pro-inflammatory cytokines IL-1β and IL-8, the anti-inflammatory milieu may explain the high frequency, prevalence and persistence of IPNV in salmon. Effects might be part of the viral mechanisms of immune evasion.

In the absence of information on functional manifestations of carotid body (CB) inflammation, we studied an experimental model in which lipopolysaccharide (LPS) administration to pentobarbitone-anaesthetized cats was performed by topical application upon the CB surface or by intravenous infusion (endotoxaemia). The latter caused: (i) disorganization of CB glomoids, increased connective tissue, and rapid recruitment of polymorphonuclear cells into the vascular bed and parenchyma within 4 h; (ii) increased respiratory frequency and diminished ventilatory chemoreflex responses to brief hypoxia (breathing 100% N(2) for 10 s) and diminished ventilatory chemosensory drive (assessed by 100% O(2) tests) during normoxia and hypoxia; (iii) tachycardia, increased haematocrit and systemic hypotension in response to LPS i.v.; and (iv) increased basal frequency of carotid chemosensory discharges during normoxia, but no change in maximal chemoreceptor responses to brief hypoxic exposures. Lipopolysaccharide-induced tachypnoea was prevented by prior bilateral carotid neurotomy. Apoptosis was not observed in CBs from cats subjected to endotoxaemia. Searching for pro-inflammatory mediators, tumour necrosis factor-alpha (TNF-alpha) was localized by immunohistochemistry in glomus and endothelial cells; reverse transcriptase-polymerase chain reaction revealed that the CB expresses the mRNAs for both type-1 (TNF-R1) and type-2 TNF-alpha receptors (TNF-R2); Western blot confirmed a band of the size expected for TNF-R1; and histochemistry showed the presence of TNF-R1 in glomus cells and of TNF-R2 in endothelial cells. Experiments in vitro showed that the frequency of carotid nerve discharges recorded from CBs perfused and superfused under normoxic conditions was not significantly modified by TNF-alpha, but that the enhanced frequency of chemosensory discharges recorded along responses to hypoxic stimulation was transiently diminished in a dose-dependent manner by TNF-alpha injections. The results suggest that the CB may operate as a sensor for immune signals, that the CB exhibits histological features of acute inflammation induced by LPS, that TNF-alpha may participate in LPS-induced changes in chemosensory activity and that some pathophysiological reactions to high levels of LPS in the bloodstream may originate from changes in CB function.

Filifolinone is a semi-synthetic terpenoid derivative obtained from Heliotropium filifolium that increases the expression level of pro-inflammatory and anti-inflammatory cytokines in kidney cells of salmon. Because cytokines are produced in response to a foreign organism and by distinct other signals modulating immune responses, we further studied the potential immunomodulatory effects of a group of structural related terpenoid derivatives from H. filifolium on salmonids to determine the relationship between the chemical structure of the derivatives and their ability to modify cytokine expression and the lymphoid content. The resin and four 3H-spiro 1-benzofuran-2,1'-cyclohexane derivatives were tested in vivo in rainbow trout (Oncorhynchus mykiss) by quantifying the transcript levels of antiviral and T helper-type cytokines and T and B cells in the kidney. Three of the four terpenoids differ only in the C-7'substituent of the cyclohexane and the presence of the ketone group at this position in Filifolinone appeared responsible of an important up-regulation of IFN-α1, IFN-γ, IL-4/13A and IL-17D in the kidney of the treated trout. In addition, the absence of a methoxy group in carbon 7 of the benzene ring, found in all compounds but not in Folifolinoic acid, produced a significant reduction of IFN-γ, IL-12 and IL-4/13A transcripts. B cells were not affected by the compound treatment but Filifolinoic acid and the resin induced a significant reduction of T cells. Altogether, results showed that immunomodulating responses observed in the trout by effect of 3H-spiro 1-benzofuran-2,1'-cyclohexane derivatives is related to the presence of the ketone group in the carbon 7' and the methoxy group in carbon 7 of the benzene ring, being Filifolinone the most active immunostimulatory compound identified.

This study reports the isolation and functional characterization of rainbow trout (Oncorhynchus mykiss) CD4-1+ T cells and the establishment of an IL-15–dependent CD4-1+ T cell line. By using Abs specific for CD4-1 and CD3ε it was possible to isolate the double-positive T cells in spleen and head kidney. The morphology and the presence of transcripts for T cell markers in the sorted CD4-1+CD3ε+ cells were studied next. Cells were found to express TCRα, TCRβ, CD152 (CTLA-4), CD154 (CD40L), T-bet, GATA-3, and STAT-1. The sorted CD4-1+ T cells also had a distinctive functional attribute of mammalian T lymphocytes, namely they could undergo Ag-specific proliferation, using OVA as a model Ag. The OVA-stimulated cells showed increased expression of several cytokines, including IFN-γ1, IL-4/13A, IL-15, IL-17D, IL-10, and TGF-β1, perhaps indicating that T cell proliferation led to differentiation into distinct effector phenotypes. Using IL-15 as a growth factor, we have selected a lymphoid cell line derived from rainbow trout head kidney cells. The morphology, cell surface expression of CD4-1, and the presence of transcripts of T cell cytokines and transcription factors indicated that this is a CD4-1+ T cell line. To our knowledge, this is the first demonstration of the presence of CD4-1+CD3ε+ T cells in salmonids. As in mammals, CD4-1+ T cells may be the master regulators of immune responses in fish, and therefore these findings and the new model T cell line developed will contribute to a greater understanding of T cell function and immune responses in teleost fish.

IFN-α1 is a type I interferon primarily produced and secreted by viral infected cells. This cytokine increases its own expression, acts as a signal to other cells and induces the expression of key proteins that antagonize virus proliferation. All nucleated cells can produce type I IFNs, although during a primary infection most of it originates from dendritic cells (DCs), especially plasmacytoid DCs (pDCs). Type I IFN genes have been described in several fish including zebrafish, channel catfish, fugu, rainbow trout and Atlantic salmon, but so far, no antibodies are available to identify the protein, evaluate expression at the protein levels or study its physiological role as antiviral cytokine. The aim of this study was to produce and characterize a monoclonal antibody against IFN-α1 of Atlantic salmon to be able to quantify the cytokine secretion and identify fish cells secreting IFN-α1. Thus, we first produced the recombinant protein (rIFN-α1) based on the published sequence of salmon IFN-α1. The gene amplified by PCR was cloned into an expression vector, E. coli BL21 (DE3) were transformed, the gene expression was induced and the protein purified. To assess the bioactivity of recombinant protein, head kidney leukocytes were stimulated in vitro for 2, 4 and 6 h with different concentrations of rIFN-α1 and the mRNA expression level of Protein Kinase RNA-activated (PKR) and myxovirus resistance gene (Mx) were analyzed. As expected, rIFN-α1 was bioactive. Then, monoclonal antibodies against rIFN-α1 were produced and three clones were selected for further characterization. The monoclonal antibody allowed us to detect IFN-α1 by ELISA and Western blot on cell supernatants and to detect IFN-α1-secreting cells by flow cytometry.

Infectious Pancreatic Necrosis Virus (IPNV) is the agent of a well-characterized acute disease that produces a systemic infection and high mortality in farmed fish species but also persistent infection in surviving fish after outbreaks. Because viral persistence of susceptible mammal hosts appears to be associated with the modulation of anti-inflammatory cytokine expression, in this study we examined the expression levels of key pro- and anti-inflammatory cytokines in kidney and spleen of trout, as well as humoral immune response (IgM and IgT) during experimental persistent viral infection and in the acute phase of infection as a comparison. IPNV infection in rainbow trout resulted in a distinct profile of cytokine expression depending on the type of infection, acute or persistent. Levels of early pro-inflammatory cytokines, IL-1β and IL-8, did not increase in the head kidney of the fish with persistent asymptomatic infection but increased in some of the symptomatic infected fish. The antiviral cytokine IFNα was not significantly induced in any of the infected fish groups. The level of expression of the Th1-related cytokine IL-12 was significantly higher in trout with persistent asymptomatic infection than in symptomatic fish. This was also accompanied by an increase in IFNγ. The anti-inflammatory cytokines IL-10 and TGF-β1 had distinct expression profiles. While IL-10 expression increased in all infected fish, TGF-β1 was only up-regulated in fish with persistent infection. All infected fish had significantly lower total IgM levels than the non-infected fish whereas IgT levels did not change. Specific and neutralizing antibodies against IPNV were not observed in acute and persistent infection except in the group of fish with the lowest degree of clinical signs. Interestingly, the lack of humoral immune response could be associated with the high expression of anti-inflammatory cytokines, which might inhibit antibody production. The balance between pro-inflammatory Th1 type cytokines and the regulatory cytokines could explain the high percentage of survival and the resolution of the inflammatory response in the IPNV-infected fish but also the establishment of viral persistence.

The immune system can be defined as a complex system that protects the organism against organisms or substances that might cause infection or disease. One of the most fascinating characteristics of the immune system is its capability to recognize and respond to pathogens with significant specificity. Innate and adaptive immune responses are able to recognize for‐ eign structures and trigger different molecular and cellular mechanisms for antigen elimina‐ tion. The immune response is critical to all individuals; therefore numerous changes have taken place during evolution to generate variability and specialization, although the im‐ mune system has conserved some important features over millions of years of evolution that are common for all species. The emergence of new taxonomic categories coincided with the diversification of the immune response. Most notably, the emergence of vertebrates coincid‐ ed with the development of a novel type of immune response. Apparently, vertebrates in‐ herited innate immunity from their invertebrate ancestors [1].

Objective To establish whether human fallopian tube (FT) epithelium can induce apoptosis in T lymphocytes and endometrial cells. Design Laboratory-based study. Setting Hospital. Patient(s) Women undergoing abdominal hysterectomy for FT samples, and women volunteers with and without endometriosis for endometrial biopsies. Intervention(s) FT samples obtained at time of surgery performed in reproductive-aged women with normal menstrual cycles. Main Outcome Measure(s) T lymphocytes or endometrial cells coincubated with FT epithelial cells and assayed for apoptosis by DNA nick-end labeling and caspase-3 activity, with the presence of Fas ligand (FasL) and Fas receptor (FasR) assessed by indirect immunostaining. Result(s) The epithelium of the FT-induced apoptosis in T cells as well as in human endometrial cells. The mechanism probably involves the FasL/FasR system; accordingly, we observed FasL at the apical surface of the epithelium and in the stroma of the FT at all phases of the menstrual cycle except during the early proliferative phase. The endometrial samples from patients with endometriosis did not express FasR and were resistant to apoptosis. Conclusion(s) In both FasR + T lymphocytes and endometrial cells, FasL + FT cells induce apoptosis. Data suggest that the FT epithelium acts as a barrier to limit the influx of lymphocytes as well as endometrial cells ascending the tube. Failure of these regulatory mechanisms may be related to the development of endometriosis.

Neisseria gonorrhoeae, the gonococcus, is a gram-negative diplococcus which causes the sexually transmitted disease gonorrhea (Figure 1). The contagious nature of gonococcal infection remains a major global health problem and represents 88 million new cases every year (WHO, 2011). N. gonorrhoeae is transmitted by human to human contact and is highly adapted to the genital tract, surviving poorly outside the human body. However, gonococcus develops resistance to antimicrobials, antigenic variability and mechanisms of immune evasion by which it evades host defenses, thus persisting and often causing undetected asymptomatic infection (Tapsall, 2001).

In vertebrates, CD3 complex and CD4 and CD8 co-receptors are essential for signal transduction during T cell activation. In the present study, we report the mRNA spliced variants of the Atlantic salmon CD3ɛ, CD4 and CD8β and the effect of pathogen encounter on the expression of these variants. CD3ɛ is alternatively spliced in thymus, head kidney, spleen and gills to give rise to the complete mRNA sequence and to an alternative product that lacks the transmembrane exon. CD4 is also alternatively spliced in the thymus, head kidney, spleen and gills to form two variants, although the alternative product is barely detectable. The alternative product lacks the exon 1B encoding the D1 domain, which is essential for binding to MHC class II proteins. Two amplicons were also found for the CD8β gene; sequencing analysis revealed that the main PCR product corresponds to the previously reported CD8β sequence, whereas the variant sequence encodes a potential protein that lacks the Ig-like domain. The expression of CD3, CD4, CD8β genes also analyzed in head kidney of LPS-treated and IPNV infected salmon and different patterns of expression were observed. The presence and balance of the different variants of T cell co-receptors could be related to the ability of fish to induce a particular type of immune response, as well as, the ability of the pathogen to modify the fish immune response.

In addition to their role in cardiorespiratory regulation, carotid body (CB) chemoreceptors serve as sensors for inflammatory status and as a protective factor during sepsis. However, lipopolysaccharide-induced sepsis (LPS) reduces CB responsiveness to excitatory or depressant stimuli. We tested whether LPS exerts a direct effect on the carotid chemoreceptor pathway, the CB and its sensory ganglion. We determined that the rat CB and nodose–petrosal–jugular ganglion complex (NPJgc) express TLR4, TNF-α and its receptors (TNF-R1 and TNF-R2). LPS administration (15 mg/kg intraperitoneally) evoked MyD88-mechanism pathway activation in CB and NPJgc, with NF-κB p65, p38 MAPK, and ERK activation. Consistently, LPS increased TNF-α and TNF-R2. Double-labeling studies showed that the aforementioned pathway occurs in TH-containing glomus cells and NPJgc neurons, components of the chemosensitive neural pathway. Thus, our results suggest that LPS acting directly through TLR4/MyD88-mechanism pathways increases TNF-α and TNF-R2 expression in the carotid chemoreceptor pathway. These results show a novel afferent pathway to the central nervous system during endotoxemia, and could be relevant in understanding sepsis pathophysiology and therapy.

Alternative splicing is an important mechanism of gene expression control that also produces a large proteome from a limited number of genes. In the immune system of mammals, numerous relevant genes have been found to undergo alternative splicing that contributes to the complexity of immune response. An increasing number of reports have recently indicated that alternative splicing also occurs in other vertebrates, such as fish. In this review we summarize the general features of such molecular events in cytokines and leukocyte co-receptors and their contribution to diversity and regulation of fish leukocytes.

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, results indicate that N. gonorrhoeae induces regulatory T cells, which might be related to the local survival of the pathogen and the establishment of a chronic asymptomatic infection.

Aging results in a general decline in the response to external insults, including acute inflammatory challenges. In young animals, the inflammatory response requires activation of the sympathetic system, including neurotransmitters such as ATP, and catecholamines (epinephrine and norepinephrine). To test whether aging affects activation of this axis, and whether this in turn might affect cytokine release, we administered lipopolysaccharide (LPS) i.p. to adult, middle-aged and aged Fisher 344 rats (6-, 15- and 23-month old, respectively) and evaluated the early (0-12h) serum levels of Neuropeptide-Y (NP-Y), ATP and vanillyl mandelic acid (VMA, as an indirect measurement of catecholamine levels). In addition, we evaluated the association between these factors and serum levels of the cytokines tumor necrosis factor-alpha (TNFalpha) and interleukin-10 (IL-10). Induction of both ATP and NP-Y was markedly reduced in the serum of aged animals, when compared to their younger counterparts, while induction of VMA was not affected by age. In spite of these changes, serum levels of TNFalpha and IL-10 were strongly hyper induced and delayed in aged rats. The results suggest that during aging there is a dysregulation in sympathetic neurotransmitter regulatory mechanisms, and this might play a role in the impairment of the inflammatory response.

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.

BACKGROUND: The factors involved in oocyte survival and transition from quiescence to the growing phenotype remain unknown. Herein we report genes that are differentially expressed in the primordial oocyte revealed by DNA arrays. METHODS: Primordial oocytes were captured selectively in rhesus monkey ovary sections using laser capture microdissection. The RNA was extracted and amplified in two rounds by T7-based linear RNA amplification, fluorescence labelled and then hybridized to human cDNA arrays containing 7680 elements. RNA from human placenta served as a reference sample. RESULTS: Ninety-five genes were found to be consistently expressed at a higher level in primordial oocytes. Expression of several of these genes in the oocyte has been reported before, e.g. deleted in azoospermia (DAZ), prohibitin and transglutaminase 2. Oocyte expression of several novel transcripts revealed on array hybridization, such as gene 33, ubiquitin-conjugating enzyme E2A, G1 to S phase transition 1, growth arrest and DNA damage-inducible (GADD), and dendritic cell-derived ubiquitin-like protein (DC-UbP) was confirmed by in situ hybridization. Some array-identified gene products [integrin b3, a-tubulin, regulatory telomere elongation protein (RAP1) and cellular repressor of EIA-stimulated genes (CREG protein)] were detected in human oocytes by immunofluorescence. Bioinformatic analysis of the oocyte-enriched transcripts reveals a functional profile summarized as follows: cell cycle (14%); transporter (13%); signal transduction (10%); cytoskeletal (7%); transcription factor (5%); immune response (5%); apoptosis-related (5%); RNA processing (5%); and the remainder of miscellaneous categories. CONCLUSIONS: These observations may contribute to the elucidation of molecular pathways involved in oocyte survival and maturation.

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.

Background Prostaglandin-E(2) and platelet-activating factor (PAF) are embryonic-derived signals that time embryo passage into the uterus in the mare and hamster respectively. PAF-like activity is detectable in the spent media of preimplantation human embryos and it has been suggested that PAF may be the embryonic signal that controls embryo transport to the uterus in our species. The actions of PAF are regulated at the level of its synthesis and degradation as well as the expression of a specific cell surface receptor (PAFr). The enzyme PAF acetylhydrolase (PAF-AH) degrades PAF. This study was undertaken to examine whether or not PAFr and PAF-AH are expressed in the human Fallopian tube and to identify the cell types in which they are expressed. Methods The presence of PAFr mRNA in tissue extracts was investigated using reverse transcription-polymerase chain reaction. We amplified the predicted amplicon for PAFr mRNA from RNA samples extracted from Fallopian tubes. The expression of PAF-AH was detected by Western blot and the localization of PAFr and PAF-AH proteins was detected by immunohistochemistry. Results Utilizing antibodies against PAFr and PAF-AH, co-localization of the two proteins in the epithelium and stromal cells were demonstrated. Conclusions These observations show that the human Fallopian tube expresses PAFr and PAF-AH at a location compatible with the proposed paracrine role of early embryo-derived PAF.

Estradiol accelerates oviductal embryo transport in the rat through changes of genomic expression in oviductal cells. However, the genes involved are unknown. We used a differential display by reverse transcription-polymerase chain reaction to detect estradiol (E2)-dependent genes in the rat oviduct. Rats on day 2 of pregnancy were untreated or treated with 10 micrograms of E2 and the oviducts were extracted at 30, 180 and 360 min later and used to isolate RNA. Products of reverse transcriptase-PCR, made with pairs of arbitrary and oligo-deoxythymidine primers, were separated on denaturing polyacrylamide gels and candidate bands were excised and reamplified. Truly positive cDNA fragments determined by a single strand conformation polymorphism assay were cloned and sequenced. A ribonuclease protection assay confirmed that clone 25 is up-regulated by E2 in the oviduct at 30, 180 and 360 min. This clone exhibited no homology with known genes and in situ hybridization showed it is only expressed in the epithelial cells of the isthmic segment. Clone 25 is likely to represent a new gene, which is up-regulated by E2 in the epithelium of the isthmic segment of the rat oviduct. Its time frame of response is compatible with a mediator of the effect of E2 on oviductal embryo transport.