A Hydrogen-Bonded Hexagonal Buckybowl Framework.

A hydrogen-bonded two-dimensionally networked buckybowl architecture is presented. Two types of hexagonal network (HexNet) structures (CPSM-1 and CPSM-2) have been achieved based on a sumanene derivative (CPSM) possessing 4,4'-dicarboxy-o-terphenyl groups in the periphery. CPSM-1 has a waved HexNet structure with an alternate alignment of upward and downward bowls. CPSM-2 has a bilayered HexNet structure composed of hamburger-shaped dimers of the bowls. This demonstrates that non-planar π-systems can be networked two-dimensionally by an appropriate supramolecular synthon to achieve structurally well-defined unique bumpy π-sheets. Furthermore, we revealed that CPSM-2 undergoes anisotropic shrinking along the c axis by 11 % under high pressure conditions (970 MPa). The shrinkage is brought about by offset sliding between bumpy π-surfaces of the bilayered HexNet sheets.

Kinetics of lymphocyte subpopulations in allogeneic grafted scales of ginbuna crucian carp.

In mammals the rejection of allografts is primarily accomplished by cell-mediated immunity including T cells. Recently, considerable studies reveal the existence of helper and cytotoxic T cell subsets in fish. Here we investigate the kinetics of CD4(+) and CD8α(+) T cells along with sIgM(+) cells and phagocytic cells in an allogeneic scale graft model using ginbuna crucian carp for understanding the mechanisms of cell-mediated immune response. The results showed that CD4(+) T cells first infiltrated into allogeneic scales followed by CD8α(+) and sIgM(+) cells, and finally phagocytic cells appeared in the graft. Furthermore, most of the CD8α(+) T cells appeared on the border of the allografted scales at the time of rejection. These results suggest that T cells play crucial roles and work together with other cell types for completion of allograft rejection.

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.

Antiviral protection mechanisms mediated by ginbuna crucian carp interferon gamma isoforms 1 and 2 through two distinct interferon gamma-receptors.

Fish genomes possess three type II interferon (IFN) genes, ifnγ1, ifnγ2 and ifnγ-related (ifnγrel). The IFNγ-dependent STAT signalling pathway found in humans and mice had not been characterized in fish previously. To identify the antiviral functions and signalling pathways of the type II IFN system in fish, we purified the ifnγ1, ifnγ2 and ifnγrel proteins of ginbuna crucian carp expressed in bacteria and found them to elicit high antiviral activities against crucian carp hematopoietic necrosis virus. We also cloned two distinct ifnγ receptor alpha chain (ifngr1) isoforms, 1 and 2, and stably expressed them in HeLa cells by transfecting the cells with ifngr1-1 or ifngr1-2 cDNA. When receptor transfectants were treated with the ligands in a one-ligand-one-receptor manner (ifnγ1 and ifngr1-2 or ifnγ2 and ifngr1-1), the stat1 protein was phosphorylated at both serine-727 and tyrosine-701 residues. Gel shift mobility analysis and reporter assay clearly showed that the specific ligand-receptor interaction resulted in the binding of the stat1 protein to the GAS element and enhanced transcription. Therefore, the actions of ifnγ1 and ifnγ2 were found to be mediated by a specific receptor for each signalling pathway via a stat1-dependent mechanism.

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 ß and CD8α in ginbuna and rainbow trout, and TCR α, ß 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.

Evaluating antigen-specific cytotoxicity of CD8+ T cells in fish by granzyme B-like activity.

Granzyme B plays an important role in granule-mediated apoptosis by CTL. It is a well characterized component of the cytolytic machinery in mammals and a candidate for the evaluation of cytotoxic activity of CTL as an alternative to conventional cytotoxicity assay. In this study, we examined the effects of granzyme inhibitors to assess the characteristics of fish granzymes in terms of substrate specificity and the involvement of granzyme B-like in the cytotoxic response. 3,4-dichloroisocoumarin (DCI), which inhibit the activity of serine protease including all members of the granzyme family, markedly suppressed the cytotoxic activity of CTL. However, CTL-mediated cytotoxicity was significantly but not completely suppressed by the addition of carbobenzyloxy-Ile-Glu-Thr-Asp-fluoromethyl ketone (Z-IETD-FMK) that specifically blocks granzyme B activity. These results suggest that additional serine proteases as well as granzyme B-like are involved in cytotoxicity of CTL in fish. We further compared cytotoxicity with the granzyme B-like hydrolytic activity against fluorogenic substrate acetyl-Ile-Glu-Thr-Asp-4-methylcoumaryl-7-amide (Ac-IETD-MCA) and found that granzyme B-like activity correlated well with the cytotoxicity of CTL in ginbuna crucian carp. Present results suggest that the granzyme activity assays is useful to assess cytotoxic activity of CTL in fish in which genetic information on granzymes and specific tools for cytotoxicity assay are not available because of well conserved catalytic triad residues and substrate binding sites in granzyme B throughout vertebrates.

Conservation of characteristics and functions of CD4 positive lymphocytes in a teleost fish.

The presence of helper and cytotoxic T cells in fish has been suggested, although T cell subsets have yet to be identified at the cellular level. In order to investigate the functions of CD4 and CD8α positive T cells we attempted to produce and characterize monoclonal antibodies (mAbs) against teleost CD4 and CD8α. Here we report the successful production of mAbs against CD4 and CD8α in clonal ginbuna crucian carp Carassius auratus langsdorfii and the function of CD4 positive T cells. In this study we demonstrate the presence of teleost CD4- and CD8α-positive T cell subsets with morphology, tissue distribution and gene expression similar to those of mammalian CD4- and CD8-positive T lymphocytes. Using mAbs we found that CD4/CD8 double positive T cells are only present in the thymus, suggesting that it is the site of T cell development. We further demonstrated in vitro proliferation of CD4 positive T cells by allogeneic combination of mixed leukocyte culture and antigen-specific proliferation of CD4 positive T cells after in vitro sensitization with OVA. In our previous study we showed that CD8α-positive lymphocytes are the primary cell type showing specific cytotoxicity against allogeneic targets. Collectively, these findings suggest that CD4 and CD8α positive T cells in ginbuna are equivalent to helper and cytotoxic T lymphocytes (CTL) in mammals, respectively. This is the first report to show the characteristics and functions of CD4 positive T cells in fish and these findings shed light into the evolutionary origins and primordial functions of helper T cells.

Clonal growth of carp (Cyprinus carpio) T cells in vitro.

Carp kidney leukocytes co-cultured with a supporting cell layer resulted in the rapid proliferation of various types of leukocytes including immature leukocytes. Expressions of marker genes for multiple blood cell lineages were observed in the primary culture. However, after several passages, the proliferating cells expressed only T cell and macrophage marker genes. Further RT-PCR analysis revealed that the proliferating cells expressed TCR constant regions (Cα, Cß, Cγ, Cδ), CD3γ/δ and CD4 (CD4L-1), but did not express CD8α and CD8ß. Additionally, in situ hybridization analysis showed that the majority of proliferating cells expressed Cα, Cß, Cγ, Cδ and CD4. Moreover, 5'-RACE sequences of TCR variable regions (Vα, Vß, Vγ, Vδ) revealed that the proliferating cells contained a polyclonal T cell repertoire, and most of the Vα and Vß sequences were functional, but the Vγ and Vδ sequences were non-functional with frame shifts and stop codons. Taken together, these results indicate that the proliferating cells after serial passages predominantly contained CD4+ CD8- αßT cells that simultaneously co-expressed non-functional γδTCR. To obtain CD4+ αßT cell (helper T cell) clones, single cells were picked up from the bulk culture, seeded into each well of 96-well plates and cultured in the presence of supporting cells and conditioned media. T cell colonies formed from single cells after 2-3 weeks. These colony cells expressed Cα, Cß, Cδ and CD4, and weakly expressed Cγ, but did not express CD8α, CD8ß and CD4L-2. Taken together, these results indicate that these clonal T cells resemble a subpopulation of mammalian CD4+ helper T cells.

Perforin-dependent cytotoxic mechanism in killing by CD8 positive T cells in ginbuna crucian carp, Carassius auratus langsdorfii.

T cell-mediated cytotoxicity occurs via pathways based on perforin or Fas mechanisms. Perforin is a protein present in the cytoplasmic granules of CD8(+) cytotoxic T lymphocytes and is secreted to form pores on target cell membranes. In fish, although the involvement of perforin in cytotoxicity have been suggested for several species, perforin-mediated cytotoxicity of CD8α(+) lymphocyte in conjunction with expression of the perforin gene has not been reported. In order to investigate the killing mechanism of CD8α(+) lymphocytes by perforin-mediated pathway in fish, we measured apoptosis of target cells triggered by CD8α(+) lymphocytes, performed cytotoxic assays in the presence or absence of perforin inhibitor; concanamycin A and EGTA, and analysed the expression of perforin1, perforin2 and perforin3 isotypic genes in ginbuna crucian carp. In the present study, we found that CTLs attached with target cells. CTL should have direct contact with target cells to kill them. Approximately 50% of target cells were positive for annexin V after co-cultured with CD8α(+) lymphocytes, indicating the induction of apoptotic cell death. Concanamycin A, which induces depolymerization of perforin resulting in lytic function, suppressed the cytotoxicity of CD8α(+) cells in a dose-dependent manner. In addition, cytotoxicity mediated by CD8α(+) lymphocytes were significantly suppressed by the addition of the Ca(2+)-chelating agents EGTA or EGTA-Mg(2+), and the addition of Ca(2+) restored the killing mechanism of target cells. We further found enhanced expression of perforin1 but not perforin2 or perforin3 in CTLs from allo-sensitized fish. The present study has demonstrated that ginbuna CTLs kill target cells through perforin-mediated pathway, suggesting that perforin-mediated pathway is conserved throughout vertebrate.

Use of flow cytometry to separate Leucocytozoon caulleryi gametocytes from avian blood.

The highly pathogenic avian protozoan Leucocytozoon caulleryi infects host chicken cells, and interference by the host genome results in difficulty in obtaining protozoal DNA for genetic analysis. We used flow cytometry analysis to separate expelled L. caulleryi gametocytes from infected chicken blood and to analyse cell populations and sorting by FACS efficiency. Infected blood cells stained with SYTO-24 showed a specific area on 2-dimensional scattergrams compared to uninfected blood. The specific area was sorted, and approximately 85% of the sorted cells were identified as L. caulleryi gametocytes by microscopic observation. DNA was also extracted from the sorted fraction, and a clear increase in polymerase chain reaction (PCR) amplification of protozoal DNA was observed compared to infected blood without sorting. Host-derived DNA was also detected by PCR; however, its amplification was decreased compared to that in unsorted infected blood. This is the first report of the separation of L. caulleryi gametocytes from infected host blood using flow cytometry. This method may be applied to further genetic analyses such as studies of the dynamics of stage-specific L. caulleryi gene expression.

A molecule in teleost fish, related with human MHC-encoded G6F, has a cytoplasmic tail with ITAM and marks the surface of thrombocytes and in some fishes also of erythrocytes.

In teleost fish, a novel gene G6F-like was identified, encoding a type I transmembrane molecule with four extracellular Ig-like domains and a cytoplasmic tail with putative tyrosine phosphorylation motifs including YxN and an immunoreceptor tyrosine-based activation motif (ITAM). G6F-like maps to a teleost genomic region where stretches corresponding to human chromosomes 6p (with the MHC), 12p (with CD4 and LAG-3), and 19q are tightly linked. This genomic organization resembles the ancestral "Ur-MHC" proposed for the jawed vertebrate ancestor. The deduced G6F-like molecule shows sequence similarity with members of the CD4/LAG-3 family and with the human major histocompatibility complex-encoded thrombocyte marker G6F. Despite some differences in molecular organization, teleost G6F-like and tetrapod G6F seem orthologous as they map to similar genomic location, share typical motifs in transmembrane and cytoplasmic regions, and are both expressed by thrombocytes/platelets. In the crucian carps goldfish (Carassius auratus auratus) and ginbuna (Carassius auratus langsdorfii), G6F-like was found expressed not only by thrombocytes but also by erythrocytes, supporting that erythroid and thromboid cells in teleost fish form a hematopoietic lineage like they do in mammals. The ITAM-bearing of G6F-like suggests that the molecule plays an important role in cell activation, and G6F-like expression by erythrocytes suggests that these cells have functional overlap potential with thrombocytes.

Kinetics of CD4+ and CD8alpha+ T-cell subsets in graft-versus-host reaction (GVHR) in ginbuna crucian carp Carassius auratus langsdorfii.

We have previously demonstrated the presence of graft-versus-host reaction (GVHR) in fish employing a model system of clonal triploid ginbuna and tetraploid ginbuna-goldfish hybrids. To elucidate the role of CD8alpha+ T cells in the induction of GVHR, we investigate the kinetics of CD4+ and CD8+ T-cell subsets in GVHR along with the pathological changes associated with GVH disease (GVHD) in ginbuna. GVHR was not induced with a leukocyte fraction lacking CD8alpha+ T cells separated by magnetic cell sorting. Ploidy and immunofluorescence analysis revealed that CD4+ and CD8alpha+ T cells from sensitized donors greatly increased in the host trunk kidney, constituting more than 80% of total cells 1-2 weeks after donor cell injection, while those from non-sensitized donors constituted less than 50% of cells present. The increase of CD4+ T cells was greater and more rapid than that of CD8alpha+ T cells. The number of donor CD4+ and CD8alpha+ T cells was highest in trunk kidney followed by spleen. Increases in donor CD4+ and CD8alpha+ T cells were also found in liver and PBL, although the percentages were not as high. Pathologic changes similar to those in human and murine acute GVHD were observed in the lymphoid organs as well as target organs such as skin, liver and intestine, including the destruction of cells and tissues and massive leukocyte infiltration. The pathologic changes became more severe with the increase of CD8alpha+ T cells. These results suggest that donor-derived CD8alpha+ T cells play essential roles for the induction of acute GVHR/D in teleosts as in mammals.

Alloantigen-specific killing is mediated by CD8-positive T cells in fish.

CD8-positive (CD8(+)) cytotoxic T lymphocytes (CTL) have antigen-specific cytotoxic activity. In fish, however, CTL expressing CD8 on their cell surface have not been identified. In order to characterize the cells involved in specific cell-mediated cytotoxicity in teleosts, we separated and sorted ginbuna kidney leucocytes into CD8alpha(+), CD4(+) and surface IgM (sIgM)(+) cells by magnetic activated cell sorting using monoclonal antibodies and examined their cytotoxic activities. Effector donor ginbuna (OB1 clone) were sensitized by allografting scales from S3N clone fish followed by injection of an allogeneic cell line (CFS) derived from S3N fish. In cytotoxic assays, target cells were labeled with CFSE and cytotoxicity was calculated based on the number of viable target cells using flow cytometry. CD8alpha(+) cells from sensitized OB1 fish showed relatively high cytotoxicity against CFS cells (immunogen) but not against allogeneic CFK cells (third party) nor isogeneic CFO cells. Pre-sensitized sIgM(+) cells exhibited cytotoxicity against not only CFS cells but also CFK cells. However, CD4(+) or CD8alpha(-) CD4(-)sIgM(-) cells as well as cells from non-sensitized fish did not show any significant cytotoxic activity. These results suggest that CD8alpha(+) cells in fish have characteristics similar to those of CTL in mammals, and that the sIgM(+) cells include NK-like cells which non-specifically killed the target cells.

In vivo characterization of primitive hematopoietic cells in clonal ginbuna crucian carp (Carassius auratus langsdorfii).

Primitive hematopoietic cells in mammalian bone marrow are purified by flow cytometry using Hoechst 33342 (Hoechst) and rhodamine-123 (Rho), because these dyes efflux activities of hematopoietic cells widely conserved in mammals. Hematopoietic stem cells (HSCs) are identified as side population (SP) cells, characterized by specific Hoechst efflux pattern in flow cytometric analysis. We previously demonstrated that SP cells from teleost body kidney (BK) had the HSC activity by a transplantation experiment using clonal ginbuna crucian carp (Carassius auratus langsdorfii). In the present study, to isolate HSCs and hematopoietic progenitor cells (HPCs) from teleosts using Hoechst and Rho, we compared the hematopoietic activity of Rho-negative (Rho(-)) cells with that of SP cells by ginbuna transplantation experiments. Rho(-) cells were clearly identified from ginbuna BK, and the majority of these cells (85%) showed a non-SP phenotype. Transplantation experiments showed that long-term repopulating activity (HSC activity) of Rho(-) cells was lower than that of SP cells, while Rho(-) cells had higher short-term repopulating activity (HPC activity) than SP cells. These results suggest that Rho(-) cells in ginbuna BK contain various stages of hematopoietic cells, while SP cells are highly enriched for HSCs, and that these dyes are useful for purification of HSCs and HPCs in teleosts.