An aromatic-dependent mutant of the fish pathogen Aeromonas salmonicida is attenuated in fish and is effective as a live vaccine against the salmonid disease furunculosis.

Aeromonas salmonicida is the etiological agent of furunculosis in salmonid fish. The disease is responsible for severe economic losses in intensively cultured salmon and trout. Bacterin vaccines provide inadequate protection against infection. We have constructed an aromatic-dependent mutant of A. salmonicida in order to investigate the possibility of an effective live-attenuated vaccine. The aroA gene of A. salmonicida was cloned in Escherichia coli, and the nucleotide sequence was determined. The codon usage pattern of aroA was found to be quite distinct from that of the vapA gene coding for the surface array protein layer (A layer). The aroA gene was inactivated by inserting a fragment expressing kanamycin resistance within the coding sequence. The aroA::Kar mutation was introduced into the chromosome of virulent A. salmonicida 644Rb and 640V2 by allele replacement by using a suicide plasmid delivery system. The aroA mutation did not revert at a detectable frequency (< 10(-11). The mutation resulted in attenuation when bacteria were injected intramuscularly into Atlantic salmon (Salmo salar L.). Introduction of the wild-type aroA gene into the A. salmonicida mutants on a broad-host-range plasmid restored virulence. A. salmonicida mutant 644Rb aroA::Kar persisted in the kidney of brown trout (Salmo trutta L.) for 12 days at 10 degrees C. Vaccination of brown trout with 10(7) CFU of A. salmonicida 644Rb aroA by intraperitoneal injection resulted in a 253-fold increase in the 50% lethal dose (LD50) compared with unvaccinated controls challenged with a virulent clinical isolate 9 weeks later. A second vaccination after 6 weeks increased the LD50 by a further 16-fold.     

Treatment of fish parasites

In research for successful treatment of fish parasitized by monogeneans, four substances (Praziquantel, Niclosamide, Levamisole-HCl, and Metrifonate) were tested in vivo against Gyrodactylus aculeati parasitizing the skin of sticklebacks (Gasterosteus aculeatus). Fish were incubated in water containing different solutions of the drugs. Praziquantel, Niclosamide and Levamisole-HCl were effective against Gyrodactylus aculeati. Praziquantel caused irreversible lesions in the parasite tegument (beginning with 1 g/ml and 90 min exposure). Niclosamide was effective in a narrow concentration range of 0.075–0.1 g/ml (90 min). Levamisole-HCl was effective in a concentration range of 20–50 g/ml for 120 min. Like Praziquantel, Niclosamide and Levamisole-HCl led to damage of the parasite tegument. The opisthaptor region and the openings of the cephalic glands were most severely affected.Furthermore, the effects of Niclosamide, Levamisole-HCl and Metrifonate on Diplozoon paradoxum parasitizing the gills of chubs (Squalius cephalus) and breams (Abramis brama) were investigated. Levamisole-HCl (10 g/ml) and Niclosamide (0.2 g/ml) were effective against Diplozoon paradoxum after an exposure of 90 min and 45 min in vitro. The parasites were severely affected along the midbody. Metrifonate caused a lysis of the tegument and a strong secretion of slime.It is suggested that chemotherapy against Gyrodactylus sp. may be accomplished with 10 mg Praziquantel/l for 3 h during storage in smaller tanks. Niclosamide (0.1 mg/l for 90 min), or Levamisole-HCl (50 mg/l for 120 min) may be used alternatively. Concentrations of Niclosamide and Levamisole-HCl have to be calculated with accuracy, since fish only tolerate a very narrow range of these drugs (Niclosamide: 0.1 mg/l for 120 min; Levamisole-HCl: 50 mg/l for 120 min).Abbreviations AC attaching clamps - AN annular depressions of the surface - BL basal lamina - BP ballon-like protrusions - BY basal labyrinth - CH central hook - CS ciliary sense cell - EV electron-pale vesicle - F fibrous content - G grooves in the tegument - IH insertion of central hooks - IN invagination of the tegument - IV inner vacuolization - LC lobes of the cephalic glands - LM longitudinal muscles - M microvillus - MI mitochondrium - MH marginal hooklet - OC openings of the cephalic glands - OH opisthaptor - P peduncle - PH prohaptor - RM ring muscles - SC surface coat - SW spider web like structure - T tegument - TR trabeculum - TS thread-like structures - V vacuole - VE vacuoles starting from electron-pale vesicles Supported by a grant of the county Nordrheinwestfalen     

Inhibition of the Aeromonas salmonicida extracellular protease by alpha 2-macroglobulin in the serum of rainbow trout

One of the major pathogenic factors of Aeromonas salmonicida, the bacterium causing furunculosis in fish, is considered to be a protease secreted in the extracellular products. This protease can be inhibited by normal trout and rabbit serum but the factor responsible has not hitherto been identified. The present results demonstrate that the A. salmonicida protease is inhibited by the alpha 2-macroglobulin of rainbow trout serum. This inhibitor accounts for about 9% of the total trypsin inhibiting capacity of trout serum, is alpha-migrating in electrophoresis, is moderately stable on storage at -20 degrees C, is destroyed by heating at 45 degrees C, has no requirement for cations and is inactivated by methylamine. The A. salmonicida protease is resistant to inhibition by 91.9% of the total trout serum trypsin inhibitors, without inactivating them, and to human alpha 1-antiproteinase. It is proposed that trout alpha 2M may have a defensive function against furunculosis but the resistance of the bacterial protease to inhibition by the majority of the serum protease inhibitors may represent pathogenic adaptation.     

Treatment of fish parasites

For chemotherapy of fish parasitized by monogeneans, a novel triazine derivative, 2-[3,5--dichloro-4-(4-methyl-sulfonylphenoxy)-phenyl]-1-methyl-hexahydro-1,2,4-triazine-3,5-dion (HOE 092 V), was tested in vivo against the gill- and skin-parasitizing speciesDactylogyrus extensus, D. vastator, andGyrodactylus arcuatus. Naturally infected fish were incubated in aerated, separate tanks at 22°C for 1,2,3, and 4 h in water containing 0, 1, 5, 10, or 15 g HOE 092 V/ml, whereasPseudodactylogyrus bini was tested in vitro at 10 g HOE 092 V/ml for 2.25 h. As seen by means of transmission electron microscopy, in vivo treatment againstD. extensus caused vacuolization and lysis of the parasite's tegument at a dose as low as 1 g/ml over a 3-h exposure period. Higher doses, such as 5 and 10 g/ml over the same exposure period, produced lesions in the circular and longitudinal musculature ofD. extensus and differing degrees of damage to the ciliary cells of protonephridia and immature vitelline cells. There was 100% mortality inD. vastator when incubation was done with 10 g HOE 092 V/ml for 4 h (G. arcuatus: 5 g/ml for 4 h; 10 g/ml for 1 h) and inP. bini after 2.25 h in vitro exposure. In all species tested, the anterior portion and the opisthaptor region were most sensitive to the drug action. This study shows that fish infected withGyrodactylus spp.,Dactylogyrus spp., and/orPseudodactylogyrus spp. can be treated successfully in a water bath containing 10 g HOE 092 V/ml.Abbreviations BL basal lamina - BM basal membrane - C caverna - CC canal cell - CI cilium - CIT cilium destroyed by treatment - CM circular musculature - CT connective tissue - DB dense body - EI electron-dense inclusion - EP electron-dense particles marking the boundary of holes - HC heterochromatin - I invagination of the tegument - IC immature vitelline cell - L lumen - LD lipid droplet - LM longitudinal musculature - LV lyzed immature vitelline cell - M mitochondrion - MC mature vitelline cell - MF myosin filament - MFI muscle fiber - MS membrane stack - N nucleus - NM nuclear membrane - OC outer membrane of cilium - OM outer membrane - P parenchyma - R ribosome - V vesicle - VA vacuole - VD vitellary droplet - VF vitelline follicle - YG Yolk globule - YL content of yolk globule     

DNA-Antiviral Vaccines: New Developments and Approaches—A Review

Current vaccines can be divided into live, recombinant and killed vaccines. Live vaccines are traditionally composed of attenuated viruses or bacteria, selected for their reduced pathogenicity. Recombinant vaccines, driven by a viral or bacterial vector express foreign antigens, or only recombinant proteins injected as antigen. Killed vaccines consist of inactivated whole pathogens. But all these traditional vaccines have some disadvantages: Attenuated live vaccine are able to undergo mutation and as mutated viruses or bacteria can now provoke the diseases against which the vaccine should protect the organism. A further disadvantage of live vaccines is the possibility of shedding which is a real problem especially in veterinary medicine. Clearly, there is a need for better vaccines to protect against diseases without the disadvantages associated with vaccines presently in use. Modern vaccines might be characterized as safe, no risk of reversion to pathogenicity, and they should be stable without the necessity of a cold chain. Production should be simple, standardized and inexpensive. Vaccine development has now been improved by the ability to use direct inoculations of plasmid DNA encoding viral or bacterial proteins. One of the major benefits of DNA-vaccines, variously termed DNA-, genetic- or nucleic acid-immunization, is the endogenous synthesis of the encoded protein. Therefore DNA vaccines mimic natural infection and provoke both strong humoral and cellular immune response. This review summarizes new developments and approaches of DNA vaccination and explains the construction of expression plasmids as well as possible mechanisms of immune responses.     

Eosinophilic granular cells (EGC) and histamine responses to Aeromonas salmonicida toxins in rainbow trout

The function of eosinophilic granular cells (EGCs) in salmonids is unknown. In a previous study of the pathogenesis of A. salmonicida, injection of crude exotoxins into rainbow trout were shown to reproduce the lesions associated with furunculosis and an accompanying lesion, the dispersion and degranulation of EGCs in the intestinal wall was reported. The present study investigated this phenomenon in relationship to the histamine content of the gut. In fish injected i/p with A. salmonicida exotoxins in a dose causing death in six hours, a coincidental decrease in the histamine content of the gut, appearance of histamine in the blood, and degranulation of the EGCs in the intestinal wall was observed 45 minutes post-injection. The fish developed behaviour patterns similar to that described by other workers for fish undergoing systemic anaphylaxis. Other features were pale gills, defaecation and widespread vasodilatation. The possible role of the EGC as a histaminogenic cell in rainbow trout is discussed.     

Interaction of the fish pathogen Aeromonas salmonicida with rainbow trout macrophages.

A procedure was developed to culture rainbow trout macrophages (M phi) on supported glass coverslips. Using this method and a variety of well-characterized Aeromonas salmonicida strains with normal or altered cell surfaces, we investigated the role of this unusual bacterial surface in the bacterium-M phi interaction. An intact crystalline protein array, the A-layer, mediated adherence of A. salmonicida cells to M phi even in the absence of opsonins. In contrast, unopsonized cells of an A-layer-negative (A-) mutant with a smooth lipopolysaccharide (LPS) layer were unable to interact with M phi. However, this ability was recovered when the A-layer was reconstituted onto the smooth LPS surface of these A- LPS+ cells. Two A. salmonicida mutants possessing the A-layer in different disorganized states had a reduced ability to interact with M phi. A+ cells grown under calcium limitation produced A-layers locked into an alternative conformation which mediated the highest levels of M phi association in the absence of opsonins or any other surface coating. Coating A+ cells with hemin greatly increased their levels of M phi association, and bacterial cells grown on trout blood agar plates also had a dramatic increase in their ability to interact with M phi. Only A+ A. salmonicida cells were highly cytotoxic to trout M phi, especially after being coated with hemin, presumably due to a more focused targeting of the bacterial cell onto the M phi surface and/or into the intracellular regions of the M phi.     

Monokine Production Following in Vitro Stimulation of the THP-1 Human Monocytic Cell Line with Pertussis Vaccine Components

Whole-cell pertussis found in diphtheria–tetanus–pertussis (DTP) vaccine can produce symptoms reminiscent of biological responses to circulating proinflammatory monokines such as IL-6, IL-1, and TNF. Therefore the ability of pertussis-containing vaccines and several heat-killed Bordetella pertussis preparations to stimulate cytokine production in a human monocytic cell line, THP-1, were examined. The whole-cell pertussis vaccine induced significantly more IL-6, IL-1, and TNF production than did the acellular pertussis or diphtheria–tetanus-only vaccine. Polymyxin B was able to inhibit most of the IL-6 induced by pertussis endotoxin and a heat-killed preparation of B. pertussis containing a null mutation in bvgAS, a regulatory locus required for expression of all known protein virulence factors synthesized by this organism. However, it only partially inhibited IL-6 production induced by other pertussis-containing preparations, including DTP vaccine. These results indicate that in vitro whole-cell vaccine is a potent stimulator of IL-6, IL-1, and TNF. They also suggest that although endotoxin is a major inducer of IL-6, other components of B. pertussis also contribute to IL-6 production by monocytes.     

Immunohistochemical studies on S-100 cells in the anterior pituitary gland of Sprague Dawley rats and spontaneous dwarf rats

The S-100 cells in the pituitary glands of adult male Sprague Dawley rats (SDs) and spontaneous dwarf rats (SDRs) were immunohistochemically examined using anti-S-100 and anti-S-100 monoclonal antibodies. The immunoreactive cells against S-100 protein were divided into three subtypes on the basis of their immunore-activity against subunits of S-100 protein: S-100 dominant type (the -type cell), S-100 dominant type (the \-type cell) and immunoreactive against both S-100 and S-100 (the -type cell). In the SD, -type cells represented 26% of the total S-100 immunoreactive cells (S-100 cells) and were localized in the peripheral area of the ventral region of the pituitary gland. This type of cell was observed forming clusters, with more abundant cytoplasm than the -type cell. The proportion of -type cells was 53%. They were diffusely distributed throughout the gland, and their processes were thicker than those of the -type cell. In the SDR, the proportion of -type cells was 55%, and they were observed throughout the gland. In contrast, -type cells totalled 12% and were localized in small areas of the central and peripheral region of the gland. The proportion of -type cells was 21% in the SD and 33% in the SDR and they were observed forming small clusters in both animal groups. The proportion of -type cells compared with the total of S-100-immunoreactive cells was significantly higher (P < 0.05) in the SDR than in the SD, while the proportion of -type cells was markedly lower (P < 0.05).     

Anticipatory postural changes induced by active unloading and comparison with passive unloading in man

Normal human subjects, sitting in a chair, were required to maintain stable elbow flexion against loads of 0.5 kg or 1.0 kg. Unloading was affected either passively by the experimenter, or actively with the subject's own contralateral arm. Elbow angle, force exerted by the load, and electromyographic activity (EMG) of biceps and triceps muscles of both arms were recorded and averaged. Passive unloading was followed by a reduction of biceps EMG activity, starting 50–80 ms after weight lift, and by an upward deflection of the forearm. With active unloading, however, a reduction of the biceps EMG activity slightly preceded the onset of unloading (0–30 ms). This reduction of the actively unloaded arm occurred at about the same time as the activity of the contralateral unloading arm. In this experiment, the unloaded forearm maintained an almost stable position. Thus, the anticipatory adjustment of elbow posture, observed when unloading was performed by the subject, appears to optimize limb stability during the mechanical perturbation.     

Cytotoxic T cells in teleost fish

The presence of antigen-specific cytotoxic T cells has been suggested in a number of in vivo and in vitro studies in fish. Acute allograft rejection with an accelerated response on second-set grafts and the presence of graft-versus-host reaction (GVHR) has been reported in teleost. Alloantigen- and virus-specific cytotoxicity has also been demonstrated in ex vivo studies in ginbuna and rainbow trout. In addition, alloantigen-specific cytotoxic T cell clones have been produced in cultures initiated with peripheral blood leukocytes (PBL) from an alloantigen-immunized channel catfish.Over the last decade several fish genomes have been sequenced and genetic information is rapidly accumulating. Thanks to these genome data bases and EST analysis, mRNA expression of T cell surface marker genes in alloantigen- or virus-specific effector cells has been reported in some fish species, e.g. TCR α or β and CD8α in ginbuna and rainbow trout, and TCR α, β or γ in channel catfish. These findings suggest the presence of CD8⁺ cytotoxic T lymphocyte (CTL) in fish similar to those of higher vertebrates. Recently, monoclonal antibodies against CD8α and CD4 antigens have been produced in some fish species. Investigation on the characteristics of CTL and cell-mediated immune mechanisms is now possible at defined T cell subsets, although identification of T cell subset is limited in a few fish species at present. In this review, we describe the recent progress in this field focusing on cells involved in antigen specific cytotoxicity.     

Cytotoxic T cells in teleost fish

The presence of antigen-specific cytotoxic T cells has been suggested in a number of in vivo and in vitro studies in fish. Acute allograft rejection with an accelerated response on second-set grafts and the presence of graft-versus-host reaction (GVHR) has been reported in teleost. Alloantigen- and virus-specific cytotoxicity has also been demonstrated in ex vivo studies in ginbuna and rainbow trout. In addition, alloantigen-specific cytotoxic T cell clones have been produced in cultures initiated with peripheral blood leukocytes (PBL) from an alloantigen-immunized channel catfish.Over the last decade several fish genomes have been sequenced and genetic information is rapidly accumulating. Thanks to these genome data bases and EST analysis, mRNA expression of T cell surface marker genes in alloantigen- or virus-specific effector cells has been reported in some fish species, e.g. TCR α or β and CD8α in ginbuna and rainbow trout, and TCR α, β or γ in channel catfish. These findings suggest the presence of CD8⁺ cytotoxic T lymphocyte (CTL) in fish similar to those of higher vertebrates. Recently, monoclonal antibodies against CD8α and CD4 antigens have been produced in some fish species. Investigation on the characteristics of CTL and cell-mediated immune mechanisms is now possible at defined T cell subsets, although identification of T cell subset is limited in a few fish species at present. In this review, we describe the recent progress in this field focusing on cells involved in antigen specific cytotoxicity.     

Paranodal Schwann cell mitochondria in spinal roots of the cat. An ultrastructural morphometric analysis

We have calculated the number of paranodal Schwann cell mitochondria in adult feline ventral and dorsal lumbar spinal roots using ultrastructural serial section analysis. Distinct accumulations of paranodal mitochondria were noted in nerve fibres more than 4-5 mm in diameter. The calculated number of paranodal mitochondria increased linearly with fibre diameter from a few hundred up to 20 000-30 000 per node. A linear increase in the number of paranodal mitochondria per node also appeared as a function of nodal variables such as nodal axon membrane area, nodal Schwann cell membrane area, and node gap extracellular volume. In large fibres (D=15-18 mm), a calculated number of about 20 000 paranodal Schwann cell mitochondria were accumulated at each node of Ranvier and related to nodal axon membrane area of about 20 mm2. Our calculations indicate that, on the average, 1000 paranodal Schwann cell mitochondria with a total volume of 6.7 mm3, a total outer membrane area of 250 mm2 and a total inner membrane area of 580 mm2 projected to each mm2 of the nodal axon membrane via the nodal Schwann cell brush border.     

Different kinds of axon terminals forming symmetric synapses with the cell bodies and initial axon segments of layer II/III pyramidal cells. I. Morphometric analysis

Summary An examination of material prepared for conventional electron microscopy has indicated that there are at least four different types of axon terminals forming symmetric synapses with the cell bodies and initial axon segments of layer II/III pyramidal cells in the rat visual cortex. One type of terminal synapses with the initial axon segment and it is derived from the chandelier cell. Because the location and features of these terminals allow them to be readily recognized, chandelier cell terminals were used to determine the extent of morphometric variability that can exist among terminals originating from one cell type. It was found that there is a wide range of mean synaptic vesicle size among chandelier terminals, so that calculated mean vesicle profile diameters for individual terminals can be between 32 and 39 run. Similar ranges of mean synaptic vesicle sizes also exist among populations of the other three axon terminal types. These terminal types are referred to as large, medium-sized, and dense terminals. The large terminals synapse with the cell bodies of layer II/III pyramids and their profiles often measure 1.5 × 0.8 m. The large terminals contain rather loosely packed pleomorphic vesicles and they frequently synapse with a second neuronal element. The medium-sized terminals are smaller, being 1.0 × 0.6–0.8 m in size, and their synaptic vesicles are usually more closely packed than those within the large terminals. The medium-sized terminals are the ones encountered most frequently on the cell bodies of pyramidal cells and they can also occur on the axon hillock and initial axon segment. The dense terminals are usually flattened against the cell body, and they contain rather rounded and closely packed synaptic vesicles, which often seem to be enmeshed in a rather dark cytoplasmic matrix. This matrix and the close packing of the vesicles makes these terminals appear to be more dense than the others. It is now necessary to determine the origins of the large, medium and dense terminals, and to ascertain if they all use GABA as their neurotransmitter.     

Integrin expression in human melanoma cells with differing invasive and metastatic properties

During the process of tumor cell invasion and metastasis, tumor cells are known to interact with extracellular matrix proteins, endothelial cells, platelets and other organ-specific structures. Integrins are cell surface molecules which mediate cell-matrix and cell-cell interactions and are likely to be important for tumor cell survival and dissemination. The purpose of this study was to characterize the integrin and proteolytic enzyme repertoire from low (A375P), medium (A375M) and high metastatic (A375SM) human melanoma cell lines. These cell lines are also invasive through human amniotic membranes in vitro and their invasiveness parallels the reported metastatic phenotype. The types and levels of expression of the various integrin receptors were analysed by quantitative immunoprecipitation using a panel of monoclonal antibodies directed to known integrin subunits. In addition, cDNA probes to the integrin subunits were used in quantitative northern blot analysis. These data show that the integrin _v3 increases 50- to 100-fold as these cells progress to a more metastatic phenotype. ₄₁ levels also appeared to increase several fold, while other ₁ integrins did not differ in their expression levels. The increased _v3 expression in the more metastatic cells resulted in an increased adhesion to vitronectin and fibrinogen substrates in cell attachment assays. However, _v- and ₃-specific antibodies did not inhibit A375 cell invasion through the amnion. Each cell line was found to release similar quantities of a 72-kDa gelatinase/type IV collagenase and tissue type plasminogen activator. These results suggest that during the progression of these tumor cells from a low to high metastatic phenotype, marked changes in integrin expression occurred which may facilitate interactions with platelets, endothelial cells and specific extracellular matrix proteins to promote metastasis.     

An electron microscopic investigation into the possible source of new muscle fibres in teleost fish

This study is based on transmission electron microscopic (TEM) investigations of deep (fast, white) teleost fish muscle proliferation in early developmental stages of three European cyprinid species and the rainbow trout. Our fine structural findings provide evidence that early myotomal growth in these animals may utilize different mechanisms that are activated in close succession during early life history. First, initial enlargement of the deep muscle bulk in the embryo seems to be due to hypertrophy of the somite-cell derived stock of muscle fibres. Second, we suggest that deep muscle growth becomes additionally powered by attachment of presumptive myogenic cells that originate from and proliferate within the adjacent mesenchymal tissue lining. Third, mesenchyme-derived muscle cell precursors are thought to enter the myotomes via the myosepta. After migration between the pre-established muscle fibres these cells may function as myosatellite cells, thus at least partly providing the stem cell population for subsequent rapid hyperplastic growth. Finally, there is evidence that presumptive deep muscle satellite cells also proliferate by mitotic division in situ. A similar process of myogenic cell migration and proliferation may foster intermediate fibre differentiation. The model of myogenic cell migration is discussed in view of in vitro and in vivo data on satellite cell migratory power and with respect to temperature-induced and species dependent differences. As for the latter, our results indicate that patterns of muscle differentiation may diverge between a fast growing salmonid species and a moderately growing cyprinid species of similar final size. The model is compatible with the wellestablished idea that teleost muscle growth may rely on different subclasses of myosatellite cells.     

Expression profiles of cytokines released in intestinal epithelial cells of the rainbow trout, Oncorhynchus mykiss, in response to bacterial infection

To determine whether fish intestinal epithelial cells (IECs) contribute to mucosal immunity, we established a method for isolating IECs from the rainbow trout Oncorhynchus mykiss and examined cytokine production in these cells. Components of the intestinal epithelium were released by incubation of intestinal pieces with 1mM dithiothreitol (DTT)/ethylenediamine tetraacetic acid (EDTA). The IEC-rich fraction (purity >90%; survival rate approximately 95%) was obtained by centrifugation on a 35%/40% Percoll gradient, followed by magnetic cell sorting using an anti-trout IgM antiserum. The gene expression profiles of 14 cytokines in trout IECs were investigated after culturing the cells for 6h with or without the pathogenic bacterium Aeromonas salmonicida. Trout IECs could produce several cytokines, of which IL-1beta and TNFalpha2 were upregulated when the cells were stimulated with live A. salmonicida. Immunohistochemical analyses with the anti-trout TNF antibody confirmed that the TNF protein was present in the IECs of trout that were intra-anally challenged with live A. salmonicida. These results show that trout IECs are an important trigger of the intestinal immune system. Further, formalin-killed A. salmonicida, conditioned medium of this bacterium, or live nonpathogenic Escherichia coli could not upregulate the expression of these cytokines. These results indicate that the production of inflammatory cytokines by IECs is caused by the adhesion of A. salmonicida, but is not due to only simple ligand-receptor interactions between the surface molecules of IECs and the bacterium or in response to bacterial secretions.     

Pronephros and spleen cells from rainbow trout (Oncorhynchus mykiss) in vitro: Growth factors produced under the influence of mitogens

Supernatants or conditioned media (CM) were produced by rainbow trout pronephros cells (PNC) and spleen cell cultures (1×10⁶/ml), by addition of 20 g/ml phythaemagglutinin (PHA), 5 ng/ml 12-O-tetradecanoyl-phorbol-13-acetate (PHA) (for 2h), PHA together with PMA, 10% horse serum (HS), or 100 g/ml concanavalin A (ConA) after a culture of 7 days. Only PNC were effective growth factor producers. The effect of different concentrations of CM (5%–28%) on cell number was tested after a cultivation period of 14 days. PNC at a concentration of 1×10⁶/ml were cultured with various concentrations of CM and the proliferation was tested by the XTT-test (testing the dehydrogenase activity of the cells by formation of a formazan) after 10 days. CM produced by cells with PHA, PHA and PMA and HS increased the proliferation in a concentration-dependent manner. CM produced with PMA alone was effective in the XTT-test. There was no synergistic or additive effect of PMA with PHA. CM produced with ConA had no effect, although in the XTT-test a strong proliferation of PNC in presence of 100 g/ml ConA was observed. In a semisolid culture with collagen, CM treatment resulted in prevention of cell death and an increase in cell size but did not induce proliferation. All CM obtained from spleen cells had no effect. Stimulation of spleen cells by CM could be seen only in the XTT-test. PHA and HS trigger the PNC to release growth factors in vitro which stimulate cell growth and/or prevent cell death.     

Synergistic activation of rat supraoptic neurosecretory neurons by noxious and hypovolemic stimuli

Summary The effects of saphenous nerve stimulation on discharge activity of supraoptic neurosecretory (NS) cells were studied in anesthetized rats. Of 112 supraoptic neurosecretory cells, 62 exhibited a phasic discharge pattern. The nerve stimulation transiently excited 46 of these 62 phasic units, as well as 35 of the 50 remaining non-phasic units. No appreciable blood pressure change was noted using PSTHs with 1-ms resolution. Though the nerve stimulation also evoked a flexor reflex of the ipsilateral hind limb, blockage of the hind limb movement with gallamine did not alter the amplitude of the supraoptic cell excitation. The threshold of the nerve stimulation was higher for the excitation than for the flexor reflex. Effects of hypovolemic and hyperosmotic stimuli on discharge activity of phasic cells during saphenous nerve stimulation were studied to find a possible interaction between these stimuli. Hemorrhage potentiated the transient excitation evoked by the nerve stimulation in all of the 8 phasic cells tested, while no such effect was seen after an injection of hypertonic sodium chloride solution in the 7 phasic cells tested. These electrophysiological data suggest that hypovolemic and noxious stimuli potentiate VP secretion in a synergistic manner but that hyperosmotic and noxious stimuli do not.Supported by grants from the Ministry of Education, Science and Culture, Japan