Adjuvant effect of allogeneic blood in vaccines against edwardsiellosis in ginbuna crucian carp Carassius auratus langsdorfii.

Edwardsiella tarda (E. tarda), an intracellular pathogen, has caused severe economic losses in aquaculture. Effective vaccine development for E. tarda prevention is urgently needed. A previous study indicates that cell-mediated immunity (CMI) might play an important role in E. tarda infection. We believe that the involvement of allograft rejection and CMI has now been well documented in mammals and some fishes. However, there is still little research on the application of blood allograft rejection in vaccine development. In the current study, we investigate the immune response and vaccine effect in fish vaccinated with allogeneic blood + formalin-killed cells vaccine (FKC), allogeneic blood + phosphate-buffered saline (PBS), PBS + FKC and PBS + PBS. In the challenge test, the relative percentage survival (RPS) of the allogeneic + FKC, the allogeneic blood + PBS and the PBS + FKC group was 61.46, 35.41, and 30.63 % respectively. The up-regulated expression of Th1-related genes IFN-γ 1, IFN-γ 1rel2, IL-12p35 and T-bet suggests the protection is via CMI induction. Only in the allogeneic + FKC group, gene expression of IFN-γ 1, IL-12p35 and T-bet is significantly higher, indicating synergy between the two substances. Furthermore, among the fish injected with the allogeneic blood cells, syngeneic blood cells and PBS group, only in the fish of the allogenic blood cells injection group, did expression of IFN-γ 1, IFN-γ 2 and IFN-γ rel2 gene expression significantly increased. The results indicate that the rejection was induced by allogeneic components. Thus, our findings might provide essential information and insights into vaccine development in aquaculture.

Local immune responses to two stages of Ichthyophthirius multifiliis in ginbuna crucian carp.

Ichthyophthirius multifiliis is a ciliated protozoan parasite and is known to infect many freshwater teleosts. Characterizing the immune system in epithelial tissues, where the parasites penetrate and settle, is key to understanding host-parasite interactions. This study examined local immune responses in vivo to the infective stage (theront and trophont) of the parasites using intra-fin administration, which has been developed to analyze in vivo immune responses using fish fin. CD8α+ and CD4+ T-cell compositions were increased significantly in the fin cavity injected with theront or trophont antigens. The expression of GATA-3 and T-bet mRNA, which regulate differentiation of helper T-cells, was upregulated significantly in leukocytes from the trophont antigen-injected site. In contrast, the percentages of macrophages and neutrophils, which are innate immunity components, were decreased significantly in the injection sites. These results suggest that I. multifiliis antigens inhibit the migration of macrophages and neutrophils, and T-cells are the first responders to I. multifiliis. Thus, to better understand the interaction of host immunity and I. multifiliis, further studies should focus on exploring the inhibitory factors from I. multifiliis or examining innate functions of teleost T-cells.

Enhancement of immune proteins expression in skin mucus of Japanese flounder Paralicthys olivaceus upon feeding a diet supplemented with high concentration of ascorbic acid.

To search immune defense proteins in skin mucus of Japanese flounder fed with a diet containing high concentration of ascorbic acid, we carried out 2D-PAGE and compared the resolved pattern of proteins between control group that fed commercial diet and ascorbic acid supplemented group (AsA group) fed a diet supplemented with high concentration of ascorbic acid (2,000 mg/kg) for 7 days. The results revealed that there were many proteins exhibited distinct increase in AsA group. Among them, 6 regions that showed a dramatic elevation were chosen for protein identification using LC-MS/MS analysis and Mascot database search. Six proteins were identified, i.e. serotransferrin (Sero), transferrin (Trans), warm temperature acclimation-related 65 kDa protein (Wap65), complement component c3 (C3), hemoglobin beta-A chain (Hbß) and apolipoprotein A-1 (Apo). Quantitative RT-PCR analysis showed that the mRNA level of Hbß in epidermis of AsA group gave much higher increase (11.6 folds) than control group; the levels of Sero/Trans, Wap65, C3 and Apo showed no apparent difference between the two groups. The mRNA levels of wap65 and c3 in the liver and Apo in the kidney of AsA group exhibited significant increase in comparison to control group. In the case of secreted immunoglobulin M (IgM) and lysozyme (lyz), no difference of the mRNA levels of IgM in epidermis, gill, kidney, spleen and intestine, and lyz in epidermis, gill, spleen and intestine, was observed. The results of in situ hybridization confirmed the elevation of Hbß mRNA level in the epidermis tissue of AsA group. Our present study provided additional evidence showing the effectiveness of AsA in activating innate immune defense system in skin mucosal tissue of fish.

Mucosal delivery of fish vaccines: Local and systemic immunity following mucosal immunisations.

The mucosal organs of fishes are directly exposed to their aquatic environment, which is suited to the colonization and growth of microorganisms, and thus these barriers are considered to play an important role in maintaining homeostasis and preventing entry of invasive pathogens. Research on fish mucosal immunity have shown that mucosal organs such as gills, skin, intestines and olfactory organs harbor lymphoid cells, including T and B cells as well as dendritic-like cells. Findings related to immune responses following direct administration of antigens into the mucosal organs could help to shed light upon the development of fish mucosal vaccines. The present review highlights vaccine delivery via mucosal organs, in particular focusing on methods other than those of typical mucosal vaccine platforms, such as oral and immersion vaccines. In addition, we propose the hypothesis that mucosal tissues are important sites for generating cell-mediated immunity following vaccination with extracellular antigens.

Availability of culture filtrate protein-10 (CFP-10) secreted from Mycobacterium pseudoshottsii for mycobacteriosis diagnosis in ginbuna crucian carp Carrasius auratus langsdorfii.

Mycobacteriosis in cultured fish is a challenge for the aquaculture industry worldwide. Treatment by chemical administration is difficult and no effective vaccine has been developed. Therefore, detection and isolation by early diagnosis are important for prevention of the spread of the disease. In mammals, interferon gamma release assays have been established for detection of tuberculosis; these tests are based on the delayed-type hypersensitivity (DTH) response against culture filtrate protein-10 (CFP-10) and the 6-kDa early secreted antigen target (ESAT-6) of Mycobacterium tuberculosis. On the other hand, little is known about the fish immune response against the ESAT-6 and CFP-10 proteins of mycobacteria, although these responses should find application in the diagnosis of mycobacteriosis in fish. In the present study, we identified ESAT-6 and CFP-10 from Mycobacterium pseudoshottsii and cloned the corresponding genes. Intraperitoneal injection of the corresponding DNA plasmid constructs in ginbuna crucian carp yielded increased expression of the fish interferon-γ1-1-encoding gene (IFN-γ1-1). In contrast, IFN-γ1-1 expression accompanied by DTH response was observed only in the CFP-10-DNA plasmid-injected fish. Furthermore, fish that had been prophylactically injected with CFP-10-DNA plasmid exhibited increased survival of M. pseudoshottsii infection. Taken together, these results suggested that CFP-10 may facilitate diagnosis of mycobacteriosis.

Sequential steps of macroautophagy and chaperone-mediated autophagy are involved in the irreversible process of posterior silk gland histolysis during metamorphosis of Bombyx mori.

To elucidate the degradation process of the posterior silk gland during metamorphosis of the silkworm ITALIC! Bombyx mori, tissues collected on the 6th day after entering the 5th instar (V6), prior to spinning (PS), during spinning (SP) and after cocoon formation (CO) were used to analyze macroautophagy, chaperone-mediated autophagy (CMA) and the adenosine triphosphate (ATP)-dependent ubiquitin proteasome. Immediately after entering metamorphosis stage PS, the levels of ATP and phosphorylated p70S6 kinase protein decreased spontaneously and continued to decline at SP, followed by a notable restoration at CO. In contrast, phosphorylated AMP-activated protein kinase α (AMPKα) showed increases at SP and CO. Most of the Atg8 protein was converted to form II at all stages. The levels of ubiquitinated proteins were high at SP and CO, and low at PS. The proteasome activity was high at V6 and PS but low at SP and CO. In the isolated lysosome fractions, levels of Hsc70/Hsp70 protein began to increase at PS and continued to rise at SP and CO. The lysosomal cathepsin B/L activity showed a dramatic increase at CO. Our results clearly demonstrate that macroautophagy occurs before entering the metamorphosis stage and strongly suggest that the CMA pathway may play an important role in the histolysis of the posterior silk gland during metamorphosis.

Recombinant carp IL-4/13B stimulates in vitro proliferation of carp IgM(+) B cells.

Teleost IL-4/13B is a cytokine related to mammalian IL-4 and IL-13, of which hitherto the function had not been studied at the protein level. We identified an IL-4/13B gene in common carp (Cyprinus carpio) and expressed the recombinant protein (rcIL-4/13B). RcIL-4/13B was shown to stimulate proliferation of IgM(+) B cells, because after four days of stimulation the IgM(+) fraction of carp kidney and spleen leukocytes had formed many cell colonies, whereas such colonies were not found in the absence of rcIL-4/13B stimulation. After nine days of incubation with rcIL-4/13B these cells had proliferated to more than 3-to-7-fold higher numbers when compared to untreated cells. The proliferating cells contained a majority of IgM(+) cells but also other cells, as indicated by FACS and RT-PCR analyses. The important conclusion is that in fish not only IL-4/13A has B cell stimulating properties, as a previous publication has shown, but also IL-4/13B.

Comparative analysis of adaptive immune response after vaccine trials using live attenuated and formalin-killed cells of Edwardsiella tarda in ginbuna crucian carp (Carassius auratus langsdorfii).

Edwardsiella tarda is an intracellular pathogen that causes edwardsiellosis in fish. Although vaccine trials with formalin-killed cells (FKC) have been reported, the vaccinations failed in protect against E. tarda infection. On the other hand, a live attenuated vaccine strategy is effective against edwardsiellosis; however, the mechanism underlying its effectiveness in fish is unclear. In the present study, we compared the adaptive immune responses in fish vaccinated with FKCs and live attenuated vaccines to elucidate the induction of adaptive immune responses following vaccination. After challenge with E. tarda, live cell (LC)-vaccinated fish showed high survival rates, high IFN-g and T-bet gene expression levels, and increased cytotoxic T lymphocytes (CTLs). In contrast, all FKC-vaccinated fish died following E. tarda infection. In addition, FKC vaccination induced high IL-4/13A and IL-10 expression levels and increased antibody titers, whereas Th1-like responses were suppressed. These results indicate that LC vaccination contributes to protection against E. tarda infection by inducing cell-mediated immunity (CMI). Thus our study findings could contribute to the development a vaccine that induces CMI against edwardsiellosis.

In vitro characteristics of cyprinid herpesvirus 2: effect of kidney extract supplementation on growth.

Herpesviral hematopoietic necrosis caused by goldfish hematopoietic necrosis virus (now identified as cyprinid herpesvirus 2, CyHV-2) has contributed to economic losses in goldfish Carassius auratus culture and is becoming a major obstacle in Prussian carp C. gibelio aquaculture in China. Several reports have described difficulties in culturing the virus, with the total loss of infectivity within several passages in cell culture. We succeeded in propagating CyHV-2 with a high infectious titer in a RyuF-2 cell line newly derived from the fin of the Ryukin goldfish variety using culture medium supplemented with 0.2% healthy goldfish kidney extract. The addition of kidney extract to the medium enabled rapid virus growth, resulting in the completion of cytopathic effect (CPE) within 4 to 6 d at 25°C. The extract also enabled reproducible virus culture with a titer of 105-6 TCID50 ml-1. The virus cultured using this protocol showed pathogenicity in goldfish after intraperitoneal injection. The virus grew in RyuF-2 cells at 15, 20, 25, 30, and 32°C but not at 34°C or higher. Higher incubation temperatures allowed earlier development of CPE, but culture at 30 and 32°C yielded a lower virus titer than that obtained at other temperatures because of heat inactivation of the propagated virus during cultivation. Cell lines derived from goldfish and ginbuna C. langsdorfii showed high susceptibility to the virus; cell lines from carp were susceptible to the virus using a medium containing goldfish kidney extract, but EPC, FHM, and BF-2 cell lines did not produce any CPE, even in the presence of the extract.

Immune responses to live and inactivated Nocardia seriolae and protective effect of recombinant interferon gamma (rIFN γ) against nocardiosis in ginbuna crucian carp, Carassius auratus langsdorfii.

Looking into the fact that substantial mortality and morbidity is associated with intracellular Gram +ve bacterium, Nocardia seriolae infection, an effective vaccine against this pathogen is necessary to control the significant losses in aquaculture practices. Therefore, an attempt was made to evaluate the effect of live (sub-lethal) and inactivated (antigenic form) N. seriolae on cellular and humoral immunity in ginbuna crucian carp, Carassius auratus langsdorfii as well as the therapeutic potency of recombinant interferon gamma (rIFN γ) against N. seriolae infection. Effect of live and inactivated N. seriolae immunisation on the proliferation of CD4(+) T cells, CD8α(+) T cells and surface Ig M(+) cells in peripheral blood leucocytes, spleen, head kidney and trunk kidney of ginbuna was studied after 1st, 3rd, 7th, 15th and 30th day post immunisation. The percentage of CD8α(+) T cells in spleen and head kidney of ginbuna was significantly higher at 3rd day post immunisation. Similarly, surface Ig M(+) cells level was found to increase in both live and inactivated N. seriolae immunised groups. On the contrary, high percentage of CD4(+) T cells was observed in live N. seriolae immunised group in both the head and trunk kidneys at 30th day post immunisation. The humoral immune response to live and inactivated N. seriolae immunised ginbuna showed high antibody titre at 15th day post immunisation but the level declined subsequently in both the immunised groups. On challenge with virulent N. seriolae (1.2 × 10(8) CFU/ml), the relative percent survival was 62.5 and 75 in live and inactivated N. seriolae immunised groups, respectively. Furthermore, we have also studied the therapeutic potency of rIFN γ and found the possible involvement of IFN γ in resistance mechanism in fish. Administration of rIFN γ into ginbuna (at 10 µg/fish) one day before challenge study was found to protect ginbuna. The relative percent survival of ginbuna was 43.75 and 60 when challenged with 2 different doses of N. seriolae i.e., 1.2 × 10(8) CFU/ml and 5 × 10(7) CFU/ml, respectively. In summary, this study indicates that both forms of N. seriolae immunisation as well as rIFN γ indeed elicit an effective protective immunity which will help in designing suitable vaccine and/or adjunct therapy against N. seriolae infection in fish.

Role of CD4(+) and CD8α(+) T cells in protective immunity against Edwardsiella tarda infection of ginbuna crucian carp, Carassius auratus langsdorfii.

Edwardsiella tarda is an intracellular pathogen that causes edwardsiellosis in fish. Our previous study suggests that cell-mediated immunity (CMI) plays an essential role in protection against E. tarda infection. In the present study, we adoptively transferred T-cell subsets sensitized with E. tarda to isogenic naïve ginbuna crucian carp to determination the T-cell subsets involved in protecting fish from E. tarda infection. Recipients of CD4(+) and CD8α(+) cells acquired significant resistance to infection with E. tarda 8 days after sensitization, indicating that helper T cells and cytotoxic T lymphocytes plays crucial roles in protective immunity to E. tarda. Moreover, transfer of sensitized CD8α(+) cells up-regulated the expression of genes encoding interferon-γ (IFN-γ) and perforin, suggesting that protective immunity to E. tarda involves cell-mediated cytotoxicity and interferon-γ-mediated induction of CMI. The results establish that CMI plays a crucial role in immunity against E. tarda. These findings provide novel insights into understanding the role of CMI to intracellular pathogens of fish.

Lack of a contact requirement for direct antibacterial activity of lymphocyte subpopulations in ginbuna crucian carp.

Cytotoxic T lymphocytes (CTL) recognize and kill cells infected with viruses, intracellular bacteria and tumors with MHC restriction and antigen specificity. In addition to these activities, recent studies in mammals have suggested that CTL can exhibit direct microbicidal activity. In our previous study we documented direct antibacterial activity of CD4(+) T cells and sIgM(+) cells as well as CD8α(+) T cells from immunized fish. However, we also found weak non-specific killing activity of lymphocytes against bacteria. In the present study we further analyzed the weak killing activity of lymphocytes, increasing the effector cell to target bacteria ratio from 10:1 to 10(3):1. Sensitized and non-sensitized effector lymphocytes (CD8α(+), CD4(+) and sIgM(+)) separated by MACS were incubated with target bacteria. CD8α(+) T cells from Edwardsiella tarda-immunized ginbuna crucian carp killed 98%, 100% and 70% of E. tarda, Streptococcus iniae and Escherichia coli, respectively. CD8α(+) T cells from non-immunized fish showed similar but slightly lower killing activity than sensitized cells. CD4(+) and sIgM(+) lymphocytes also showed high killing activity against E. tarda and S. iniae as found for CD8α(+) T cells, although the activity was lower against E. coli. Supernatants from all three types of lymphocytes showed microbicidal activity, although the activity was lower than that evoked by effector lymphocytes. Furthermore, the presence of a membrane between effectors and targets did not affect the killing activity. The present results suggest that both sensitized and non-sensitized lymphocytes non-specifically killed target bacteria without the need of contact. The major difference between the present and previous experiments is the E:T ratio. We suspect that there are two different mechanisms in the direct bacterial killing by lymphocytes in ginbuna.

Transcription analysis of two Eomesodermin genes in lymphocyte subsets of two teleost species.

Eomesodermin (Eomes), a T-box transcription factor, is a key molecule associated with function and differentiation of CD8(+) T cells and NK cells. Previously, two teleost Eomes genes (Eomes-a and -b), which are located on different chromosomes, were identified and shown to be expressed in zebrafish lymphocytes. For the present study, we identified these genes in rainbow trout and ginbuna crucian carp. Deduced Eomes-a and -b amino acid sequences in both fish species contain a highly conserved T-box DNA binding domain. In RT-PCR, both Eomes transcripts were readily detectable in a variety of tissues in rainbow trout and ginbuna. The high expression of Eomes-a and -b in brain and ovary suggests involvement in neurogenesis and oogenesis, respectively, while their expression in lymphoid tissues presumably is associated with immune functions. Investigation of separated lymphocyte populations from pronephros indicated that both Eomes-a and -b transcripts were few or absent in IgM(+) lymphocytes, while relatively abundant in IgM(-)/CD8α(+) and IgM(-)/CD8α(-) populations. Moreover, we sorted trout CD8α(+) lymphocytes from mucosal and non-mucosal lymphoid tissues and compared the expression profiles of Eomes-a and -b with those of other T cell-related transcription factor genes (GATA-3, T-bet and Runx3), a Th1 cytokine gene (IFN-γ) and a Th2 cytokine gene (IL-4/13A). Interestingly, the tissue distribution of Eomes-a/b, T-bet, and Runx3 versus IFN-γ transcripts did not reveal simple correlations, suggesting tissue-specific properties of CD8α(+) lymphocytes and/or multiple modes that drive IFN-γ expressions.

Characterization of fish-specific IFNγ-related binding with a unique receptor complex and signaling through a novel pathway.

Unlike mammals, fish express two type II interferons, IFNγ and fish-specific IFNγ (IFNγ-related or IFNγrel). We previously reported the presence of two IFNγrel genes, IFNγrel 1 and IFNγrel 2, which exhibit potent antiviral activity in the Ginbuna crucian carp, Carassius auratus langsdorfii. We also found that IFNγrel 1 increased allograft rejection; however, the IFNγrel 1 receptor(s) and signaling pathways underlying this process have not yet been elucidated. In this study, we examined the unique signaling mechanism of IFNγrel 1 and its receptors. The phosphorylation and transcriptional activation of STAT6 in response to recombinant Ginbuna IFNγrel 1 (rgIFNγrel 1) was observed in Ginbuna-derived cells. Binding of rgIFNγrel 1 to Class II cytokine receptor family members (Crfbs), Crfb5 and Crfb17, which are also known as IFNAR1 and IFNGR1-1, respectively, was detected by flow cytometry. Expression of the IFNγrel 1-inducible antiviral gene, Isg15, was highest in Crfb5- and Crfb17-overexpressing GTS9 cells. Dimerization of Crfb5 and Crfb17 was detected by chemical crosslinking. The results indicate that IFNγrel 1 activates Stat6 through an interaction with unique pairs of receptors, Crfb5 and Crfb17. Indeed, this cascade is distinct from not only that of IFNγ but also that of known IFNs in other vertebrates. IFNs may be classified by their receptor and signal transduction pathways. Taken together, IFNγrel 1 may be classified as a novel type of IFN family member in vertebrates. Our findings provide important information on interferon gene evolution in bony fish.

Direct antibacterial activity of CD8+/CD4+ T-cells in ginbuna crucian carp, Carassius auratus langsdorfii.

Cytotoxic T cells (CTLs) constitute an important component of the specific effector mechanism in killing against microbial-infected or transformed cells. In addition to these activities, recent studies in mammals have suggested that CTLs can exhibit direct antimicrobial activity. Therefore, the present investigation was conducted to find out the microbicidal activity of CD8α(+) T cells of ginbuna crucian carp, Carassius auratus langsdorfii. The CD8α(+) T cells from immunised ginbuna exhibited the antibacterial activity against both facultative intracellular bacteria and extracellular bacteria. The maximum reduction of viable count of pathogens was recorded with effector (sensitized) cells and target (bacteria) ratio of 10:1 co-incubated for a period of 1-2 h at 26 °C when effector cells were derived from ginbuna 7 days after one booster dose at 15th day of primary sensitization/immunisation. Sensitized CD8α(+) T cells are found to kill 92.1 and 98.9% of Lactococcus garvieae and Edwardsiella tarda, respectively. No significant difference in the bacterial killing activity could be recorded against facultative intracellular bacteria and extracellular bacteria. The specificity study indicated the non-specific killing of bacteria. CD8α(+) T cells from E. tarda immunised ginbuna exhibited 40% of non-specific killing activity against L. garvieae and those from L. garvieae immunised ginbuna showed 42.7% of non-specific killing activity against E. tarda. Furthermore, CD4(+) T cells also killed 88% and 95.7% of L. garvieae and E. tarda, respectively. In addition to T cell subsets, surface IgM(+) cells also killed both types of pathogens. Therefore, the present study demonstrated the direct antibacterial activity of CD8α(+), CD4(+) T-cells and surface IgM(+) cells in fish.

Teleost T and NK cell immunity.

The main function of the immune system is to maintain the organism's homeostasis when invaded by foreign material or organisms. Prior to successful elimination of the invader it is crucial to distinguish self from non-self. Most pathogens and altered cells can be recognized by immune cells through expressed pathogen- or danger-associated molecular patterns (PAMPS or DAMPS, respectively), through non-self (e.g. allogenic or xenogenic cells) or missing major histocompatibility (MHC) class I molecules (some virus-infected target cells), and by presenting foreign non-self peptides of intracellular (through MHC class I-e.g. virus-infected target cells) or extracellular (through MHC class II-e.g. from bacteria) origin. In order to eliminate invaders directly or by destroying their ability to replicate (e.g. virus-infected cells) specialized immune cells of the innate and adaptive responses appeared during evolution. The first line of defence is represented by the evolutionarily ancient macrophages and natural killer (NK) cells. These innate mechanisms are well developed in bony fish. Two types of NK cell homologues have been described in fish: non-specific cytotoxic cells and NK-like cells. Adaptive cell-mediated cytotoxicity (CMC) requires key molecules expressed on cytotoxic T lymphocytes (CTLs) and target cells. CTLs kill host cells harbouring intracellular pathogens by binding of their T cell receptor (TCR) and its co-receptor CD8 to a complex of MHC class I and bound peptide on the infected host cell. Alternatively, extracellular antigens are taken up by professional antigen presenting cells such as macrophages, dendritic cells and B cells to process those antigens and present the resulting peptides in association with MHC class II to CD4(+) T helper cells. During recent years, genes encoding MHC class I and II, TCR and its co-receptors CD8 and CD4 have been cloned in several fish species and antibodies have been developed to study protein expression in morphological and functional contexts. Functional assays for innate and adaptive lymphocyte responses have been developed in only a few fish species. This review summarizes and discusses recent results and developments in the field of T and NK cell responses with focus on economically important and experimental model fish species in the context of vaccination.

Clonal growth of carp (Cyprinus carpio) T cells in vitro: long-term proliferation of Th2-like cells.

Carp kidney leukocytes co-cultured with a supporting cell layer resulted in proliferation of polyclonal CD4(+) αßT cells as described previously. These bulk-cultured T cells expressed transcripts for both T helper 1 cells (Th1) master regulator (T-bet) and T helper 2 cells (Th2) master regulator (GATA-3). To identify the Th subsets in bulk-cultured T cells, single cells were picked up from the bulk culture, proliferated, and characterized. The majority of the clones displayed characteristics consistent with CD4(+) αßT cell identity. These clones expressed both TCRα and TCRß, but could not produce a TCRγδ heterodimer since they typically only expressed either TCRγ or TCRδ. These clones also expressed the TCR co-receptor genes CD4-1 or CD4-2, whereas they did not express CD8α or CD8ß. In addition, GATA-3 was expressed whereas T-bet was not. Among these clones, one clone (KoThL5) continued to proliferate on the supporting cells and was successively transferred for more than 10 months and 90-100 passages. To characterize the KoThL5 cells by their cytokine production profile, they were stimulated with PHA and investigated by real-time RT-PCR. mRNA expression of Th2-related cytokine (IL-4/13B) was only enhanced in KoThL5 cells whereas both Th1-related cytokine (IFNγ) and Th2-related cytokines (IL-4/13A and IL-4/13B) were significantly enhanced in bulk-cultured T cells. Taken together, KoThL5 cells share some features with mammalian Th2 cells. This is the first study to describe in vitro cultures of teleost cell with Th2-like features. The KoThL5 cell line has considerable potential for addressing questions concerning the properties of teleost Th2 cells.

Comparative gene expression analysis of zebrafish and mammals identifies common regulators in hematopoietic stem cells.

Hematopoiesis in teleost fish is maintained in the kidney. We previously reported that Hoechst dye efflux activity of hematopoietic stem cells (HSCs) is highly conserved in vertebrates, and that Hoechst can be used to purify HSCs from teleost kidneys. Regulatory molecules that are strongly associated with HSC activity may also be conserved in vertebrates. In this study, we identified evolutionarily conserved molecular components in HSCs by comparing the gene expression profiles of zebrafish, murine, and human HSCs. Microarray data of zebrafish kidney side population cells (zSPs) showed that genes involved in cell junction and signal transduction tended to be up-regulated in zSPs, whereas genes involved in DNA replication tended to be down-regulated. These properties of zSPs were similar to those of mammalian HSCs. Overlapping gene expression analysis showed that 40 genes were commonly up-regulated in these 3 HSCs. Some of these genes, such as egr1, gata2, and id1, have been previously implicated in the regulation of HSCs. In situ hybridization in zebrafish kidney revealed that expression domains of egr1, gata2, and id1 overlapped with that of abcg2a, a marker for zSPs. These results suggest that the overlapping genes identified in this study are regulated in HSCs and play important roles in their functions.

Induction of both local and systemic immunity by in vivo injection of PHA into ginbuna carp fin.

Phytohemagglutinin (PHA) is a well-known mitogen inducing activation and proliferation of lymphocytes, particularly T lymphocytes in vitro. PHA has also been used in vivo for assessing cell-mediated immunity in non-mammalian vertebrates, particularly in birds. However, it has been suggested that local inflammation as a direct result of tissue damage could be responsible for skin swelling after PHA injection, in addition to induction of T lymphocyte mitogenesis. In order to understand the complex nature of this response in fish we investigated the accumulation of cell types chronologically in dorsal fin of ginbuna crucian carp Carassius auratus langsdorfii after PHA injection. Neutrophils appeared first and showed a peak response on day 1, decreasing gradually and followed by macrophages and blast cells while lymphocytes increased later with a peak response on day 5. The number of accumulated cells was significantly higher in PHA-injected fish than controls in most cases. Lymphocytes identified as CD4-1+and CD8α+ were significantly more abundant in PHA-injected fish than in control fish throughout the 7-day experimental period except on day 1, while the number of IgM+ lymphocytes was higher in PHA-injected fish only on day 1. In the blast cell fraction, the number of CD4-1+ lymphocytes was significantly higher in PHA-injected fish than in control fish throughout experimental period, except on day 1. We also document the migration of neutrophils from the kidney to the fin through blood, followed by granulopoiesis in the kidney. These results suggest that adaptive immunity as well as innate immunity was induced by in vivo stimulation with PHA.