Avances en medicina regenerativa

Además de los autotrasplantes de médula ósea para tratamiento de leucemias y de piel para regeneración de quemaduras, existen nuevas técnicas y avances clínicos muy recientes en medicina regenerativa. Los principales protagonistas son las células madre, las automedicinas vivas del siglo XXI

A sandwich ELISA to detect VHSV and IPNV in turbot

Abstract: The recent demonstration that reared turbot (Scophthalmus maximus L) is a natural host for salmonid rhabdoviruses has made their rapid detection relevant to these fish species. A unique protocol to select and use non-competitive monoclonal antibodies (Mabs) for two high-sensitivity sandwich ELISAs has been developed to detect both infectious pancreatic necrosis virus (IPNV) and viral haemorrhagic septicaemia virus (VHSV) in turbot kidney extracts to assess the possibility of using them in field diagnosis. For maximum sensitivity, turbot kidney extracts can be two-fold diluted with high-ionic strength buffers and assayed for the presence of the major viral proteins (VMS rhabdovirus nucleoprotein N/Nx and/or IPN birnavirus protein VP3). The use of control plates coated with irrelevant mouse antibodies (IgG1 and IgG2a) in parallel ELISAs allows for a precise estimation of possible false positives. Turbot kidney extracts with low levels of virus might now be assayed directly without using cell culture, with high precision and in a short time during the acute phase of these viral diseases in reared turbot.Acuidoro, SL; INI

Rainbow trout surviving infections of viral haemorrhagic septicemia virus (vhsv) show lasting antibodies to recombinant g protein fragments

P. 929-935Rainbow trout antibodies (Abs) binding to recombinant fragments (frgs) derived from the protein G of the viral haemorrhagic septicemia virus (VHSV)-07.71 strain, could be detected by ELISA (frg-ELISA) in sera from trout surviving laboratory-controlled infections. Abs were detected not only by using sera from trout infected with the homologous VHSV isolate but also with the VHSV-DK-201433 heterologous isolate, which had 13 amino acid changes. Sera from healthy trout and/or from trout surviving infectious haematopoietic necrosis virus (IHNV) infection, were used to calculate cut-off absorbances to differentiate negative from positive sera. Specific anti-VHSV Abs could then be detected by using any of the following frgs: frg11 (56-110), frg15 (65-250), frg16 (252-450) or G21-465. While high correlations were found among the ELISA values obtained with the different frgs, no correlations between any frg- ELISA and complement-dependent 50 % plaque neutralization test (PNT) titres could be demonstrated. Between 4 to 10 weeks after VHSV-infection, more trout sera were detected as positives by using heterologous frg-ELISA rather than homologous PNT. Furthermore, the percentage of positive sera detected by frg11-ELISA increased with time after infection to reach 100 %, while those detected by complement-dependent PNT decreased to 29.4 %, thus confirming that the lack of neutralising Abs does not mean the lack of any anti-VHSV Abs in survivor trout sera. Preliminary results with sera from field samples suggest that further refinements of the frg-ELISA could allow detection of anti-VHSV trout Abs in natural outbreaks caused by different heterologous VHSV isolates.The homologous frg-ELISA method could be useful to follow G immunization attempts during vaccine development and/or to best understand the fish Ab response during VHSV infections. The viral frgs approach might also be used with other fish species and/or viruses.S

Rainbow Trout Red Blood Cells Exposed to Viral Hemorrhagic Septicemia Virus Up-Regulate Antigen-Processing Mechanisms and MHC I&II, CD86, and CD83 Antigen-presenting Cell Markers

Nucleated teleost red blood cells (RBCs) are known to express molecules from the major histocompatibility complex and peptide-generating processes such as autophagy and proteasomes, but the role of RBCs in antigen presentation of viruses have not been studied yet. In this study, RBCs exposed ex vivo to viral hemorrhagic septicemia virus (VHSV) were evaluated by means of transcriptomic and proteomic approaches. Genes and proteins related to antigen presentation molecules, proteasome degradation, and autophagy were up-regulated. VHSV induced accumulation of ubiquitinated proteins in ex vivo VHSV-exposed RBCs and showed at the same time a decrease of proteasome activity. Furthermore, induction of autophagy was detected by evaluating LC3 protein levels. Sequestosome-1/p62 underwent degradation early after VHSV exposure, and it may be a link between ubiquitination and autophagy activation. Inhibition of autophagosome degradation with niclosamide resulted in intracellular detection of N protein of VHSV (NVHSV) and p62 accumulation. In addition, antigen presentation cell markers, such as major histocompatibility complex (MHC) class I & II, CD83, and CD86, increased at the transcriptional and translational level in rainbow trout RBCs exposed to VHSV. In summary, we show that nucleated rainbow trout RBCs can degrade VHSV while displaying an antigen-presenting cell (APC)-like profil

Rag1 immunodeficiency‐induced early aging and senescence in zebrafish are dependent on chronic inflammation and oxidative stress [Poster]

12th European Zebrafish Meeting, Krakow, 9-13 July 2023In mammals, recombination activating gene 1 (RAG1) plays a crucial role in adaptive immunity, generating a vast range of immunoglobulins. Rag1−/− zebrafish (Danio rerio) are viable and reach adulthood without obvious signs of infectious disease in standard nonsterile conditions, suggesting that innate immunity could be enhanced to compensate for the lack of adaptive immunity. By using microarray analysis, we confirmed that the expression of immunity- and apoptosis-related genes was increased in the rag1−/− fish. This tool also allows us to notice alterations of the DNA repair and cell cycle mechanisms in rag1−/− zebrafish. Several senescence and aging markers were analyzed. In addition to the lower lifespan of rag1−/− zebrafish compared to their wild-type (wt) siblings, rag1−/− showed a higher incidence of cell cycle arrest and apoptosis, a greater amount of phosphorylated histone H2AX, oxidative stress and decline of the antioxidant mechanisms, an upregulated expression and activity of senescence-related genes and senescence-associated β-galactosidase, respectively, diminished telomere length, and abnormal self-renewal and repair capacities in the retina and liver. Metabolomic analysis also demonstrated clear differences between wt and rag1−/− fish, as was the deficiency of the antioxidant metabolite L-acetylcarnitine (ALCAR) in rag1−/− fish. Therefore, Rag1 activity does not seem to be limited to V(D)J recombination but is also involved in senescence and aging. Furthermore, we confirmed the senolytic effect of ABT-263, a known senolytic compound and, for the first time, the potential in vivo senolytic activity of the antioxidant agent ALCAR, suggesting that this metabolite is essential to avoid premature agingN

Induction of colony formation by phytohemagglutinin stimulation of rainbow trout kidney in fibrin cultures

The cells from rainbow trout kidney, its main hematopoietic organ, are able to proliferate by forming colonies in fibrin-clots in the presence of phytohemagglutinin (a mitogen of mammalian T lymphocytes). The addition of phytohemagglutinin induced colonies formed by lymphocytes, eccentric-nuclei cells, multinucleated cells and large nucleated cells. The heterogeneity of this response suggests that, in this primitive animal the immunological reactions could be simpler than in mammals, one unique mitogen giving rise to proliferation of more than one cellular type. This method could be applied to the study of the immune response in the case of viral or bacterial infections, in trouts as well as in other animals including men

Fish mass immunization against virus with recombinant “spiny” bacterins

Bacterins obtained from recombinant bacteria displaying heterologous antigens in its surface coded by prokaryotic rather than eukaryotic expression plasmids (here called “spiny” bacterins or spinycterins), have been used to increase fish immunogenicity of recombinant viral protein fragments. To explore their immunogenicity, five bacterial-specific membrane anchor-motifs characterized in the literature (Nmistic, Mistic, NTD, YAIN and YBEL) were genetically fused to the immunorelevant cystein-free 117 amino acid fragment II from the ORF149 of cyprinid herpes virus 3 (frgIICyHV3). The fusion of anchor-motifs to the N-terminus of frgIICyHV3 enriched expression in E.coli outer membranes as demonstrated by ELISA, immunofluorescence and flow cytometry of formaldehyde-fixed recombinant bacteria (spinycterins). Unconventional low-intensity ultrasound inducing mucosal micropores in a reversible non-harmful manner was used before carp or zebrafish immersion on spinycterin suspensions as a practical delivery alternative to fish-to-fish injection. After ELISA screening for anti-frgIICyHV3-specific antibodies of spinycterin-immunized fish plasma, the YBEL constructs were identified as the most immunogenic in both carp and zebrafish, correlating with one of the best expressed recombinant proteins as demonstrated by Western blot and surface enriched as demonstrated by ELISA and flow cytometry. The use of prokaryotic expression plasmids to express viral immunorelevant protein fragments in traditionally used fish vaccination bacterins should reduce the environmental concerns raised by DNA vaccination based on eukaryotic expression plasmids. Therefore, spinycterins may be a useful alternative to develop safer fish viral vaccines and mass vaccination methods. © 201

Inhibition of SERPINe1 reduces rhabdoviral infections in zebrafish

While exploring the molecular mechanisms behind the fin hemorrhages that follow zebrafish (Danio rerio) early infection with viral haemorrhagic septicemia virus (VHSV), we discovered that most serpin (serine protease inhibitor) gene transcripts were upregulated, except those of serpine1. Surprisingly, only SERPINe1. -derived 14-mer peptide and low molecular weight drugs targeting SERPINe1 (i.e. tannic acid, EGCG, tiplaxtinin) inhibited in vitro infections not only of VHSV, but also of other fish rhabdoviruses such as infectious hematopoietic necrosis virus (IHNV) and spring viremia carp virus (SVCV). While the mechanisms that inhibited rhabdoviral infections remain speculative, these and other results suggested that SERPINEe1-derived peptide specifically targeted viral infectivity rather than virions. Practical applications might be developed from these studies since preliminary evidences showed that tannic acid could be used to reduce VHSV-caused mortalities. These studies are an example of how the identification of host genes targeted by viral infections using microarrays might facilitate the identification of novel prevention drugs in aquaculture and illuminate viral infection mechanisms. © 2015 Elsevier Ltd

Techniques for diagnosing viral diseases of salmonid fish

Cell culture for amplification and the techniques most used for identificaiton of salmonid viruses - neutralization, immunofluorescence and, to a lesser extent, immunoperoxidase, complement fixation, agglutination, electron microscopy, immunodiffusion or radioimmunoassay - may soon be replaced by other techniques such as enzyme immunoassays (immunodot and enzyme-linked immunosorbent assay, ELISA) and hybridization with DNA probes. It is expected that developments in monoclonal antibodies and amplification by the polymerase chain reaction will increase sensitivity of enzyme immunoassays and DNA hybridizations, respectively. -from Author

One-step purification of the major rainbow trout immunoglobulin

Salmonid immunoglobulin purification has been hampered by time-consuming, labour-intensive, conventional chromatographic procedures. In this report we describe the use of immunoaffinity chromatography for the purification of immunoglobulins from trout serum in a single step. An anti-heavy chain monoclonal antibody (1G7) which reacts with more than 85% of total trout immunoglobulins was used to purify the trout immunoglobulin. The purity achieved was higher than 95%, and the immunoglobulins recovered were fully immunogenic. This method served equally well in isolating immunoglobulins from the sera of other salmonids (coho salmon and chinook salmon). The procedure should be useful in the standardization of salmonid immunoglobulins reagents. © 1991