A retrospective study suggests 55 days of persistence of SARS-CoV-2 during the first wave of the pandemic in Santiago de Chile.

Background: As the COVID-19 pandemic persists, infections continue to surge globally. Presently, the most effective strategies to curb the disease and prevent outbreaks involve fostering immunity, promptly identifying positive cases, and ensuring their timely isolation. Notably, there are instances where the SARS-CoV-2 virus remains infectious even after patients have completed their quarantine. Objective: Understanding viral persistence post-quarantine is crucial as it could account for localized infection outbreaks. Therefore, studying and documenting such instances is vital for shaping future public health policies. Design: This study delves into a unique case of SARS-CoV-2 persistence in a 60-year-old female healthcare worker with a medical history of hypertension and hypothyroidism. The research spans 55 days, marking the duration between her initial and subsequent diagnosis during Chile's first COVID-19 wave, with the analysis conducted using RT-qPCR. Results: Genomic sequencing-based phylogenetic analysis revealed that the SARS-CoV-2 detected in both Nasopharyngeal swab samples (NPSs) was consistent with the 20B clade of the Nextstrain classification, even after a 55-day interval. Conclusion: This research underscores the need for heightened vigilance concerning cases of viral persistence. Such instances, albeit rare, might be pivotal in understanding sporadic infection outbreaks that occur post-quarantine.

Importance of Probiotics in Fish Aquaculture: Towards the Identification and Design of Novel Probiotics.

Aquaculture is a growing industry worldwide, but it faces challenges related to animal health. These challenges include infections by parasites, bacteria, and viral pathogens. These harmful pathogens have devastating effects on the industry, despite efforts to control them through vaccination and antimicrobial treatments. Unfortunately, these measures have proven insufficient to address the sanitary problems, resulting in greater environmental impact due to the excessive use of antimicrobials. In recent years, probiotics have emerged as a promising solution to enhance the performance of the immune system against parasitic, bacterial, and viral pathogens in various species, including mammals, birds, and fish. Some probiotics have been genetically engineered to express and deliver immunomodulatory molecules. These promote selective therapeutic effects and specific immunization against specific pathogens. This review aims to summarize recent research on the use of probiotics in fish aquaculture, with a particular emphasis on genetically modified probiotics. In particular, we focus on the advantages of using these microorganisms and highlight the main barriers hindering their widespread application in the aquaculture industry.

Analysis of microbiota-host communication mediated by butyrate in Atlantic salmon.

Butyrate is a microbiota-produced metabolite, sensed by host short-chain fatty acid receptors FFAR2 (Gpr43), FFAR3 (Gpr41), HCAR2 (Gpr109A), and Histone deacetylase (HDAC) that promotes microbiota-host crosstalk. Butyrate influences energy uptake, developmental and immune response in mammals. This microbial metabolite is produced by around 79 anaerobic genera present in the mammalian gut, yet little is known about the role of butyrate in the host-microbiota interaction in salmonid fish. To further our knowledge of this interaction, we analyzed the intestinal microbiota and genome of Atlantic salmon (Salmo salar), searching for butyrate-producing genera and host butyrate receptors. We identified Firmicutes, Proteobacteria, and Actinobacteria as the main butyrate-producing bacteria in the salmon gut microbiota. In the Atlantic salmon genome, we identified an expansion of genes orthologous to FFAR2 and HCAR2 receptors, and class I and IIa HDACs that are sensitive to butyrate. In addition, we determined the expression levels of orthologous of HCAR2 in the gut, spleen, and head-kidney, and FFAR2 in RTgutGC cells. The effect of butyrate on the Atlantic salmon immune response was evaluated by analyzing the pro and anti-inflammatory cytokines response in vitro in SHK-1 cells by RT-qPCR. Butyrate decreased the expression of the pro-inflammatory cytokine IL-1ß and increased anti-inflammatory IL-10 and TGF-ß cytokines. Butyrate also reduced the expression of interferon-alpha, Mx, and PKR, and decreased the viral load at a higher concentration (4 mM) in cells treated with this molecule before the infection with Infectious Pancreatic Necrosis Virus (IPNV) by mechanisms independent of FFAR2, FFAR3 and HCAR2 expression that probably inhibit HDAC. Moreover, butyrate modified phosphorylation of cytoplasmic proteins in RTgutGC cells. Our data allow us to infer that Atlantic salmon have the ability to sense butyrate produced by their gut microbiota via different specific targets, through which butyrate modulates the immune response of pro and anti-inflammatory cytokines and the antiviral response.

Genomic Evidence Suggests Viral Persistence of SARS-CoV-2 for 386 Days in Health Worker: A Case Report from Santiago of Chile.

The COVID-19 pandemic continues to affect several countries. One of the best ways to control its spread is the timely identification of infected patients for isolation and quarantine. While an episode of infection lasts an average of 8-10 days from the onset of symptoms, there is literature describing long-lasting viral persistence events. Here, we report a case of persistence of SARS-CoV-2 for 386 days in a health worker from Santiago de Chile. Our study could be one of the longest reported viral persistence events. RNA sequencing analyses indicated that the first positive diagnosis (8 June 2020) corresponded to a SARS-CoV-2 variant belonging to Clade Nextstrain 20A. Three hundred eighty-six days later (23 September 2021), the second positive result reached the same viral variant (Clade 20A) but without presence or circulation in Chile since May 2021. Both sequencing coverages showed an identity of 99.21%, with some mutations related to the severity of the disease (ORF1b:P314L) and more infectivity (S:D614G). This work reinforces the idea of implementing an RT-qPCR or rapid antigen test once the quarantine is fulfilled to ensure viral absence, identify potential persistence, and, consequently, minimize the risk of local outbreaks of SARS-CoV-2 infection.

Infectious pancreatic necrosis virus (IPNV) recombinant viral protein 1 (VP1) and VP2-Flagellin fusion protein elicit distinct expression profiles of cytokines involved in type 1, type 2, and regulatory T cell response in rainbow trout (Oncorhynchus mykiss).

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-ß1 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+ T cells suggesting that Th differentiation happens in response to VP1. This is also consistent with the expression of t-bet and gata3, the master regulators for Th1/Th2 differentiation in the kidneys of immunized animals. A different cytokine expression profile was found after VP2-Flg administration, i.e., upregulation occurs for ifn-γ, il-4/13a, il-10a, and tgf-ß1, while down-regulation was observed in il-4/13b2 and il-2a. The cytokine response was due to flagellin; only the il-2a effect was dependent upon VP2 in the fusion protein. To the best of our knowledge this study reports for the first-time characteristics of the adaptive immune response induced in response to IPNV VP1 and the fusion protein VP2-Flg in fish. VP1 induces cytokines able to trigger the humoral and cell-mediated immune response in rainbow trout. The analysis of the fish response against VP2-Flg revealed the immunogenic properties of Aeromonas salmonicida flagellin, which can be further tested for adjuvanticity. The novel immunogenic effects of VP1 in rainbow trout open new opportunities for further IPNV vaccine development using this viral protein.

Comparison of the First and Second Wave of Infections by SARS-CoV-2: A Retrospective and Longitudinal Study From a Primary Health Care Center in Santiago of Chile.

The current COVID-19 pandemic is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Many countries have reported the experience of at least two contagion waves, describing associated mortality rates and population behavior. The analysis of the effect of this pandemic in different localities can provide valuable information on the key factors to consider in the face of future massive infectious diseases. This work describes the first retrospective and comparative study about behavior during the first and second waves of the COVID-19 pandemic in Chile from a primary Healthcare Center. From 19,313 real-time quantitative PCR (RT-qPCR) tests assessed, the selected 1,694 positive diagnostics showed a decrease in mortality rate in the second wave (0.6%) compared with the first (4.6%). In addition, we observed that infections in the second wave were mainly in young patients with reduced comorbidities. The population with a complete vaccination schedule shows a decrease in the duration of symptoms related to the disease, and patients with more comorbidities tend to develop severe illness. This report provides evidence to partially understand the behavior and critical factors in the severity of the COVID-19 pandemic in the population of Santiago of Chile.

First Identification of Reinfection by a Genetically Different Variant of SARS-CoV-2 in a Homeless Person from the Metropolitan Area of Santiago, Chile.

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.

Variations in Rainbow Trout Immune Responses against A. salmonicida: Evidence of an Internal Seasonal Clock in Oncorhynchus mykiss.

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 (Oncorhynchus mykiss) kept under constant photoperiod and water temperature against intraperitoneally injected Aeromonas salmonicida during the summer and winter were investigated. The kinetics of different leukocyte subpopulations from peritoneal cavity, spleen, and head kidney in response to the bacteria was measured by flow cytometry. Furthermore, the kinetics of induced A. salmonicida-specific antibodies was evaluated by ELISA. Despite maintaining the photoperiod and water temperature as constant, different cell baselines were detected in all organs analyzed. During the winter months, B- and T-cell responses were decreased, contrary to what was observed during summer months. However, the specific antibody titers were similar between the two seasons. Natural antibodies, however, were greatly increased 12 h post-injection only during the wintertime. Altogether, our results suggest a bias toward innate immune responses and potential lymphoid immunosuppression in the wintertime in trout. These seasonal differences, despite photoperiod and water temperature being kept constant, suggest an internal inter-seasonal or circannual clock controlling the immune system and physiology of this teleost fish.

Revisiting the Teleost Thymus: Current Knowledge and Future Perspectives.

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.

The Atlantic salmon interleukin 4/13 receptor family: Structure, tissue distribution and modulation of gene expression.

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.

Single-Nucleotide Polymorphisms (SNP) Mining and Their Effect on the Tridimensional Protein Structure Prediction in a Set of Immunity-Related Expressed Sequence Tags (EST) in Atlantic Salmon (Salmo salar).

Single-nucleotide polymorphisms (SNPs) are single genetic code variations considered one of the most common forms of nucleotide modifications. Such SNPs can be located in genes associated to immune response and, therefore, they may have direct implications over the phenotype of susceptibility to infections affecting the productive sector. In this study, a set of immune-related genes (cc motif chemokine 19 precursor [ccl19], integrin ß2 (itß2, also named cd18), glutathione transferase omega-1 [gsto-1], heat shock 70 KDa protein [hsp70], major histocompatibility complex class I [mhc-I]) were analyzed to identify SNPs by data mining. These genes were chosen based on their previously reported expression on infectious pancreatic necrosis virus (IPNV)-infected Atlantic salmon phenotype. The available EST sequences for these genes were obtained from the Unigene database. Twenty-eight SNPs were found in the genes evaluated and identified most of them as transition base changes. The effect of the SNPs located on the 5'-untranslated region (UTR) or 3'-UTR upon transcription factor binding sites and alternative splicing regulatory motifs was assessed and ranked with a low-medium predicted FASTSNP score risk. Synonymous SNPs were found on itß2 (c.2275G > A), gsto-1 (c.558G > A), and hsp70 (c.1950C > T) with low FASTSNP predicted score risk. The difference in the relative synonymous codon usage (RSCU) value between the variant codons and the wild-type codon (ΔRSCU) showed one negative (hsp70 c.1950C > T) and two positive ΔRSCU values (itß2 c.2275G > A; gsto-1 c.558G > A), suggesting that these synonymous SNPs (sSNPs) may be associated to differences in the local rate of elongation. Nonsynonymous SNPs (nsSNPs) in the gsto-1 translatable gene region were ranked, using SIFT and POLYPHEN web-tools, with the second highest (c.205A > G; c484T > C) and the highest (c.499T > C; c.769A > C) predicted score risk possible. Using homology modeling to predict the effect of these nonsynonymous SNPs, the most relevant nucleotide changes for gsto-1 were observed for the nsSNPs c.205A > G, c484T > C, and c.769A > C. Molecular dynamics was assessed to analyze if these GSTO-1 variants have significant differences in their conformational dynamics, suggesting these SNPs could have allosteric effects modulating its catalysis. Altogether, these results suggest that candidate SNPs identified may play a crucial potential role in the immune response of Atlantic salmon.

Interleukin (IL)-2 Is a Key Regulator of T Helper 1 and T Helper 2 Cytokine Expression in Fish: Functional Characterization of Two Divergent IL2 Paralogs in Salmonids.

Mammalian interleukin (IL)-2 is a cytokine centrally involved in the differentiation and survival of CD4+ T helper subsets and CD4+ T regulatory cells and in activation of cytotoxic effector lymphocytes. In bony fish, IL2 orthologs have been identified with an additional divergent IL2-Like gene on the same locus present in several fish species. We report here two divergent IL2 paralogs, IL2A and IL2B, in salmonids that originated from the whole genome duplication event in this fish lineage. The salmonid IL2 paralogs differ not only in sequence but also in exon sizes. The IL-2 isoforms that are encoded have disparate pI values and may have evolved to preferentially bind specific IL-2 receptors. Rainbow trout IL2 paralogs are highly expressed in thymus, spleen, gills, kidney and intestine, important tissues/organs in fish T cell development and function. Their expression in peripheral blood leukocytes (PBL) is low constitutively but can be upregulated by the mixed leukocyte reaction, by the T cell mitogen phytohemagglutinin and by signal mimics of T cell activation (phorbol 12-myristate 13-acetate and calcium ionophore). Both trout IL-2 isoforms promoted PBL proliferation and sustained high-level expression of CD4 and CD8, suggesting that trout IL-2 isoforms are T cell growth/survival factors mainly expressed by activated T cells. The recombinant proteins for these two trout IL2 paralogs have been produced in E. coli and possess shared but also distinct bioactivities. IL-2A, but not IL-2B, induced IL12P35A1 and CXCR1 expression in PBL. IL-2B had a stronger effect on upregulation of the T helper 1 (Th1) cytokine interferon-γ (IFNγ) and could sustain CD8α and CD8ß expression levels. Nevertheless, both cytokines upregulated key Th1 (IFNγ1, IFNγ2, TNFα2 and IL12) and T helper 2 (Th2) cytokines (IL4/13B1 and IL4/13B2), cytokine and chemokine receptors and the antimicrobial peptide cathelicidin-1 but had limited effects on T helper 17 cytokines and TGFß1 in PBL. They could also enhance PBL phagocytosis. These results suggest, for the first time in fish, that IL-2 isoforms may have an important role in regulating Th1 and Th2 cell development, and innate and adaptive host defenses in fish, and shed light on lineage-specific expansion, evolution, and functional diversification of IL2 in vertebrates.

Characterisation of rainbow trout peripheral blood leucocytes prepared by hypotonic lysis of erythrocytes, and analysis of their phagocytic activity, proliferation and response to PAMPs and proinflammatory cytokines.

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-1ß and IL-6). Furthermore, the PBL can respond to stimulation with PAMPs (LPS, poly I:C) and cytokines (IL-1ß 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.

Synergistic interactions between phenolic compounds identified in grape pomace extract with antibiotics of different classes against Staphylococcus aureus and Escherichia coli.

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 ß-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.

Vaccines for piscirickettsiosis (salmonid rickettsial septicaemia, SRS): the Chile perspective.

INTRODUCTION: Piscirickettsia salmonis (P. salmonis) is the aetiological bacterium of the contagious disease piscirickettsiosis or salmonid rickettsial septicaemia (SRS) and causes significant economic losses to aquaculture production in Chile. Current strategies to control infection are i) indiscriminate antibiotic use and ii) vaccination with predominantly P. salmonis bacterin vaccines that do not provide acceptable levels of protection against piscirickettsiosis. Areas covered: This review covers the basic biology of P. salmonis, clinical piscirickettsiosis and disease control, the development of current P. salmonis vaccines, innate and adaptive immunity and a 5-year plan to develop new piscirickettsiosis vaccines. Expert commentary: Fundamental knowledge is lacking on the complexities of P. salmonis-host interactions, relating to bacterial virulence and host innate and adaptive immune responses, which needs to be addressed. The development of new P. salmonis vaccines needs the application of comprehensive 'omics' technologies to identify candidate vaccine antigens capable of stimulating long-lasting protective immune responses.

Isolation and Characterization of Salmonid CD4+ T Cells.

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, TCRß, 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-ß1, 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.

Key cytokines of adaptive immunity are differentially induced in rainbow trout kidney by a group of structurally related geranyl aromatic derivatives.

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.

Induction of anti-inflammatory cytokine expression by IPNV in persistent infection.

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-1ß 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-ß1 had distinct expression profiles. While IL-10 expression increased in all infected fish, TGF-ß1 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.

Neisseria gonorrhoeae induces a tolerogenic phenotype in macrophages to modulate host immunity.

Neisseria gonorrhoeae is the etiological agent of gonorrhoea, which is a sexually transmitted disease widespread throughout the world. N. gonorrhoeae does not improve immune response in patients with reinfection, suggesting that gonococcus displays several mechanisms to evade immune response and survive in the host. N. gonorrhoeae is able to suppress the protective immune response at different levels, such as B and T lymphocytes and dendritic cells. In this study, we determined whether N. gonorrhoeae directly 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- ß 1) but not the production of proinflammatory cytokine TNF- α , indicating that gonococcus induces a shift towards anti-inflammatory cytokine production. Moreover, N. gonorrhoeae did not induce significant upregulation of costimulatory CD86 and MHC class II molecules. We also showed that N. gonorrhoeae infected 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 that N. gonorrhoeae induces a tolerogenic phenotype in antigen-presenting cells, which seems to be one of the mechanisms to induce evasion of immune response.

Fas ligand(+) fallopian tube epithelium induces apoptosis in both Fas receptor(+) T lymphocytes and endometrial cells.

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.