Jaime Pignatelli

Early immune responses in rainbow trout liver upon viral hemorrhagic septicemia virus (VHSV) infection.

Among the essential metabolic functions of the liver, in mammals, a role as mediator of systemic and local innate immunity has also been reported. Although the presence of an important leukocyte population in mammalian liver is well documented, the characterization of leukocyte populations in the teleost liver has been only scarcely addressed. In the current work, we have confirmed the presence of IgM+, IgD+, IgT+, CD8α+, CD3+ cells, and cells expressing major histocompatibility complex (MHC-II) in rainbow trout (Oncorhynchus mykiss) liver by flow cytometry and/or immunohistochemistry analysis. Additionally, the effect of viral hemorrhagic septicemia virus (VHSV) on the liver immune response was assessed. First, we studied the effect of viral intraperitoneal injection on the transcription of a wide selection of immune genes at days 1, 2 and 5 post-infection. These included a group of leukocyte markers genes, pattern recognition receptors (PRRs), chemokines, chemokine receptor genes, and other genes involved in the early immune response and in acute phase reaction. Our results indicate that T lymphocytes play a key role in the initial response to VHSV in the liver, since CD3, CD8, CD4, perforin, Mx and interferon (IFN) transcription levels were up-regulated in response to VHSV. Consequently, flow cytometry analysis of CD8α+ cells in liver and spleen at day 5 post-infection revealed a decrease in the number of CD8α+ cells in the spleen and an increased population in the liver. No differences were found however in the percentages of B lymphocyte (IgM+ or IgD+) populations. In addition, a strong up-regulation in the transcription levels of several PRRs and chemokines was observed from the second day of infection, indicating an important role of these factors in the response of the liver to viral infections.

Identification of Teleost Skin CD8α + Dendritic-like Cells, Representing a Potential Common Ancestor for Mammalian Cross-Presenting Dendritic Cells

Although fish constitute the most ancient animal group in which an acquired immune system is present, the presence of dendritic cells (DCs) in teleosts has been addressed only briefly, and the identification of a specific DC subset in teleosts remained elusive because of the lack of specific Abs. In mice, DCs expressing CD8α+ in lymphoid tissues have the capacity to cross-present extracellular Ags to T cells through MHC I, similarly to tissue-derived CD103+ DCs and the human CD141+ DC population. In the current study, we identified a large and highly complex subpopulation of leukocytes coexpressing MHC class II and CD8α. This CD8α+ MHC II+ DC-like subpopulation constituted ∼1.2% of the total leukocyte population in the skin, showing phenotypical and functional characteristics of semimature DCs that seem to locally regulate mucosal immunity and tolerance in a species lacking lymph nodes. Furthermore, we identified trout homologs for CD141 and CD103 and demonstrated that, in trout, this skin CD8+ DC-like subpopulation expresses both markers. To our knowledge, these results provide the first evidence of a specific DC-like subtype in nonimmune tissue in teleosts and support the hypothesis of a common origin for all mammalian cross-presenting DCs.

Transcriptional heterogeneity of IgM+ cells in rainbow trout (Oncorhynchus mykiss) tissues.

Two major classes of B lymphocytes have been described to date in rainbow trout: IgM+ and IgT+ cells. IgM+ cells are mainly localized in the spleen, peripheral blood and kidney but are also found in other tissues. However, differences among IgM+ cell populations attending to its location are poorly defined in fish. Thus, the aim of this work was to characterize the expression of different immune molecules such as chemokine receptors, Toll-like receptors (TLRs) and transcription factors on sorted IgM+ lymphocytes from different rainbow trout tissues. IgM+ populations from blood, spleen, kidney, gills, intestine and liver were isolated by cell sorting and the constitutive levels of transcription of these genes evaluated by real-time PCR. To further characterize B cells, we identified an MS4A sequence. In humans, the MS4A family includes several genes with immune functions, such as the B cell marker CD20 or FcRβ. Subsequently, we have also evaluated the mRNA levels of this MS4A gene in the different IgM+ populations. The relevant differences in transcriptional patterns observed for each of these IgM+ populations analyzed, point to the presence of functionally different tissue-specific B cell populations in rainbow trout. The data shown provides a pattern of genes transcribed in IgM+ B cells not previously revealed in teleost fish. Furthermore, the constitutive expression of all the TLR genes analyzed in IgM+ cells suggests an important role for these cells in innate immunity.

A Global Transcriptome Analysis Reveals Molecular Hallmarks of Neural Stem Cell Death, Survival, and Differentiation in Response to Partial FGF-2 and EGF Deprivation

Neurosphere cell culture is a commonly used model to study the properties and potential applications of neural stem cells (NSCs). However, standard protocols to culture NSCs have yet to be established, and the mechanisms underlying NSC survival and maintenance of their undifferentiated state, in response to the growth factors FGF-2 and EGF are not fully understood. Using cultures of embryonic and adult olfactory bulb stem cells (eOBSCs and aOBSCs), we analyzed the consequences of FGF-2 and EGF addition at different intervals on proliferation, cell cycle progression, cell death and differentiation, as well as on global gene expression. As opposed to cultures supplemented daily, addition of FGF-2 and EGF every 4 days significantly reduced the neurosphere volume and the total number of cells in the spheres, mainly due to increased cell death. Moreover, partial FGF-2 and EGF deprivation produced an increase in OBSC differentiation during the proliferative phase. These changes were more evident in aOBSC than eOBSC cultures. Remarkably, these effects were accompanied by a significant upregulation in the expression of endogenous Fgf-2 and genes involved in cell death and survival (Cryab), lipid catabolic processes (Pla2g7), cell adhesion (Dscaml1), cell differentiation (Dscaml1, Gpr17, S100b, Ndrg2) and signal transduction (Gpr17, Ndrg2). These findings support that a daily supply of FGF-2 and EGF is critical to maintain the viability and the undifferentiated state of NSCs in culture, and they reveal novel molecular hallmarks of NSC death, survival and the initiation of differentiation.

Molecular characterization of CD9 and CD63, two tetraspanin family members expressed in trout B lymphocytes

Tetraspanins are a family of membrane-organizing proteins, characterized by the presence of four highly conserved transmembrane regions that mediate diverse physiological functions. In the current study, we have identified two novel tetraspanin members in rainbow trout (Oncorhynchus mykiss), homologs to mammalian CD9 and CD63. Both genes were expressed in muscle, skin, gills, hindgut, gonad, liver, spleen, head kidney, thymus and peripheral blood leukocytes. Throughout the early life cycle stages, CD9 mRNA levels significantly increased after first feeding, whereas CD63 transcription remained constant during all the developmental stages analyzed. In response to an experimental bath infection with viral hemorrhagic septicemia virus (VHSV), CD9 transcription was down-regulated in the gills, while CD63 mRNA levels were down-regulated in the head kidney. Instead, when the virus was intraperitoneally injected, the transcription of both genes was significantly up-regulated in peritoneal cells at several days post-infection. Additionally, both genes were transcriptionally up-regulated in the muscle of trout injected with a VHSV DNA vaccine. To gain insight on the relation of these tetraspanins with B cell activity we determined their constitutive expression in naive IgM(+) populations from different sources and observed that both molecules were being transcribed by IgM(+) cells in different tissues. Furthermore, CD9 transcription was significantly down-regulated in splenic IgM(+) cells in response to in vitro VHSV exposure. Our results provide insights on the potential role of these tetraspanins on teleost B cell and antiviral immunity.

Nurr1 blocks the mitogenic effect of FGF-2 and EGF, inducing olfactory bulb neural stem cells to adopt dopaminergic and dopaminergic-GABAergic neuronal phenotypes.

The transcription factor Nurr1 is expressed in the mouse olfactory bulb (OB), although it remains unknown whether it influences the generation of dopaminergic neurons (DA) (DA neurons) in cells isolated from this brain region. We found that expressing Nurr1 in proliferating olfactory bulb stem cells (OBSCs) produces a marked inhibition of cell proliferation and the generation of immature neurons immunoreactive for tyrosine hydroxylase (TH) concomitant with marked upregulations of Th, Dat, Gad, and Fgfr2 transcripts. In long‐term cultures, these cells develop neurochemical and synaptic markers of mature‐like mesencephalic DA neurons, expressing GIRK2, VMAT2, DAT, calretinin, calbindin, synapsin‐I, and SV2. Concurring with the increase in both Th and Gad expression, a subpopulation of induced cells was both TH‐ and GAD‐immunoreactive indicating that they are dopaminergic‐GABAergic neurons. Indeed, these cells could mature to express VGAT, suggesting they can uptake and store GABA in vesicles. Remarkably, the dopamine D1 receptor agonist SKF‐38393 induced c‐Fos in TH+ cells and dopamine release was detected in these cultures under basal and KCl‐evoked conditions. By contrast, cotransducing the Neurogenin2 and Nurr1 transcription factors produced a significant decrease in the number of TH‐positive neurons. Our results indicate that Nurr1 overexpression in OBSCs induces the formation of two populations of mature dopaminergic neurons with features of the ventral mesencephalon or of the OB, capable of responding to functional dopaminergic stimuli and of releasing dopamine. They also suggest that the accumulation of Fgfr2 by Nurr1 in OBSCs may be involved in the generation of DA neurons.

Transcriptional Regulation of Olfactory Bulb Neurogenesis

The neurons in the olfactory bulb originate from molecularly defined and spatially distinct proliferative regions. Glutamatergic projection neurons are generated during the embryonic period in the local ventricular zone of the olfactory bulb, a territory in the dorsal telencephalon in which the transcription factor Pax6 is expressed. Some cells in this zone also express Tbr1, a marker of glutamatergic neurons. By contrast, embryonic olfactory bulb interneurons are derived from Gsx2 expressing cells in the dorsal lateral ganglionic eminence of the ventral telencephalon, and from progenitors outside the dorsal lateral ganglionic eminence, including the olfactory bulb neuroepithelium. Postnatally, interneurons arise from the subventricular zone of the lateral ventricle, although the rostral migratory stream and the olfactory bulb also appear to serve as a source of neurons. Transcription factors are crucial to generate all classes of neurons and glia in the olfactory bulb, both during development and adulthood. In this article, we discuss and propose models on how the spatial and temporal regulation of transcription factor expression controls the self‐renewal, proliferation and cell fate of neural stem cells and progenitors, which finally leads to the generation of distinct functional subtypes of neurons in the developing and adult olfactory bulb. Anat Rec, 296:1364‐1382, 2013.

Characterization of BAFF and APRIL subfamily receptors in rainbow trout (Oncorhynchus mykiss). Potential role of the BAFF / APRIL axis in the pathogenesis of proliferative kidney disease

Proliferative kidney disease (PKD) is a parasitic infection of salmonid fish characterized by hyper-secretion of immunoglobulins in response to the presence of the myxozoan parasite, Tetracapsuloides bryosalmonae. In this context, we hypothesized that the BAFF/APRIL axis, known to play a major role in B cell differentiation and survival in mammals, could be affected by the parasite and consequently be involved in the apparent shift in normal B cell activity. To regulate B cell activity, BAFF and APRIL bind to transmembrane activator and calcium modulator and cyclophilin ligand interactor (TACI) and B cell maturation antigen (BCMA), whereas BAFF also binds to BAFF receptor (BAFF-R). In teleost fish, although some BAFF and APRIL sequences have been reported, their receptors have not been identified. Thus, as a first step in the current work, we have identified homologues to mammalian TACI, BCMA and BAFF-R in rainbow trout (Oncorhynchus mykiss), that constitute the first report of BAFF and APRIL receptor sequences in fish. Subsequently we studied the transcriptional modulation of BAFF, APRIL, and the fish-specific related cytokine, BALM and their putative receptors in fish naturally exposed to T. bryosalmonae. Finally, to gain further insights on the functional role that these cytokines play during the course of PKD, we have studied their effect on the survival of kidney IgM+ B cells and on immunoglobulin transcription. Our results support the premise that the BAFF / APRIL axis could play an important role during PKD, which may open the possibility of new therapeutic treatments against the disease.

Seroprevalence of porcine torovirus (PToV) in Spanish farms

BackgroundTorovirus infections have been associated with gastroenteritis and diarrhea in horses, cows, pigs and humans, especially in young animals and in children. Although asymptomatic in a large percentage of cases, however toroviruses may pose a potential threat to worsen disease outcome in concurrent infections with other enteric pathogens. Previous studies based on the analysis of limited numbers of samples indicated high seroprevalences against porcine torovirus (PToV) in various European countries. The aim of this work was to perform a seroepidemiological survey of PToV in Spanish farms in order to define the seroprevalence against this virus.ResultsSerum samples (n = 2664) from pigs of different ages were collected from 100 Spanish farms coming from 10 regions that concentrate 96.1% of the 3392 farms with 80 or more sows censused in Spain. Samples were screened by means of an indirect enzyme-linked immune-sorbent assay (ELISA) based on a recombinant PToV nucleocapsid protein as antigen. The analysis of the whole serum collection yielded a total of 95.7% (2550/2664) seropositive samples. The highest prevalence (99.6%, 1382/1388) and ELISA values (average O.D. ± standard deviation) were observed in the sows (1.03±0.36) and the lowest prevalence (59.4%, 98/165) and anti-PToV IgG levels (0.45±0.16) were found amongst 3-week-old piglets. Both ELISA reactivity values and seroprevalence percentages rose quickly with piglet’s age from 3 to 11 weeks of age; the seroprevalence was 99.3% (2254/2270) when only the samples from sows and pigs over 11-weeks of age were considered. Antibodies against PToV were detected in all analyzed farms.ConclusionsThis report describes the results of the largest torovirus seroepidemiological survey in farmed swine performed so far. Overall, the seroprevalence against PToV in animals older than 11 weeks of age was >99%, indicating that this virus is endemic in pig herds from Spain.

New Insights on the Structure and Morphogenesis of Berne Virus

torovirus morphogenesis is still poorly understood and further studies are required to characterize this process at both morphological and molecular levels as well as to understand the structure of torovirus particles. All the information about the structure and morphogenesis of toroviruses comes from early studies performed by conventional electron microscopy examination of thin sections from cultured equine dermis cells infected with BEV, as well as from intestinal tissue from calves infected with the bovine torovirus BRV. These studies provided detailed descriptions of the different viral assemblies. However, in the past two decades new methods for structural analysis by