Transfer of Immunity to Tumour Isografts by the Systemic Administration of Xenogeneic “Immune” RNA

SPECIFIC immunoreactivity can be conferred on lymphoid cells by incubation with RNA-rich extracts prepared from lymphoid tissues exposed to specific antigens in vivo¹ and in vitro^2,3. We have shown transfer of immunity to tumour specific antigens in vivo⁴ and in vitro⁵ by incubation of syngeneic spleen cells in vitro with RNA extracted from the lymphoid tissues of xenogeneic or syngeneic animals immunized with the tumour to be treated. Administration of these spleen cells to normal animals decreased the development and growth of isografts of the same tumour.     

Responsiveness of Thymus Cells to Syngeneic and Allogeneic Lymphoid Cells

THE thymus is necessary for the normal development of cell-mediated immunity in mice as shown by the immunological defects after neonatal thymectomy¹. Thymus cells themselves can be stimulated by allogeneic lymphoid cells in mixed leucocyte reaction (MLR)² and become killer cells or cytotoxic lymphocytes after stimulation with allogeneic spleen cells in vitro (H. Wagner and M. Feldmann, unpublished work) and in vivo^3,4. This suggests that the thymus as well as peripheral lymphoid tissues contain T cells which can be stimulated by foreign histocompatibility antigen to divide and differentiate into the cytotoxic lymphocytes which mediate cellular immunity. There have been suggestions that thymus cells might be stimulated to divide by “self” antigen, as well as foreign cells: incorporation of ³H-thymidine above background levels has been found in cultures with syngeneic spleen and thymus cells of adult rats⁵, although the experiments do not determine whether thymus or spleen cells have been stimulated. In contrast to these experiments, Howe et al. reported that only thymus cells of neonatal CBA mice reacted to allogeneic and syngeneic spleen cells of adult animals in “one way” MLR cultures^6,7. Whether the reaction of neonatal thymus cells to syngeneic adult spleen cells is recognition of “self” antigens is uncertain, since spleens of adult mice could carry antigens which do not occur in neonatal animals and are therefore “unknown” for neonatal thymus cells. We demonstrate here that neonatal thymus cells do not react to 4-day-old CBA spleen cells, but adult thymus cells do react against both allogeneic and syngeneic adult spleen cells.     

Reversible Blocking Effect of Anti-mouse Immunoglobulin Serum on the Induction of Immunity and Tolerance <Emphasis Type="Italic">in vitro</Emphasis>

THE induction of blast transformation by incubating lymphocytes with anti-immunoglobulin¹ and anti-allotype² sera has suggested that these cells have immunoglobulin on their surface. This hypothesis was directly verified by the demonstration of immunoglobulin on living mouse lymphoid cells by Raff et al.³. There is much evidence to indicate that immunocompetent cells have surface receptors for antigen. This idea is based on the finding that lymphocytes can bind radioactively labelled antigen to their surface^4,5 and that specific immune unresponsiveness occurs if lymphoid cells are exposed to either highly radioactive antigen⁶ or haptens capable of forming covalent bonds with proteins^7,8. The immunoglobulin nature of these antigen receptors is suggested by recent work showing that the binding of radioactively labelled antigen can be blocked by anti-immunoglobulin sera^5,9. Reports that the adoptive immune response of mouse spleen cells can be inhibited by anti-mouse immunoglobulin sera (AMS)^9,10 suggest that the interaction of antigen with the immunoglobulin receptor sites is a crucial step in the induction of the antibody response. We report here that the inhibitory action of AMS on the immune response is potentially reversible and that the induction of immune tolerance to polymerized flagellin (POL) in vitro may be blocked in the presence of AMS.     

Immunoreactivity in a Cellular Immune Reaction of Lymphocytes preabsorbed on Cell Monolayers

SELECTIVE removal from non-immune lymphoid cell populations, of lymphocytes involved ultimately in production of antibody against haptenic determinants, has been demonstrated^1,2 using the particular haptenic group coupled to an inert substrate. In a series of preliminary experiments, we have used monolayer cultures of fibroblast cells to remove lymphocytes, from a population of non-immune lymph node cells, capable of reacting against cellular antigens represented in the monolayer culture. Our test system in these experiments is the in vitro graft reaction model of Ginsburg³ as modified by Berke et al.⁴.     

Role of PKCtheta in macrophage-mediated immune response to <Emphasis Type="Italic">Salmonella typhimurium</Emphasis> infection in mice

Background

The serine/threonine protein kinase C (PKC) theta has been firmly implicated in T cell-mediated immunity. Because its role in macrophages has remained undefined, we employed PKCtheta-deficient (PKCtheta ^?/?) mice in order to investigate if PKCtheta plays a role in macrophage-mediated immune responses during bacterial infections.

Results

Our results demonstrate that PKCtheta plays an important role in host defense against the Gram-negative, intracellular bacterium Salmonella typhimurium, as reflected both by markedly decreased survival and a significantly enhanced number of bacteria in spleen and liver of PKCtheta ^?/? mice, when compared to wild-type mice. Of note, albeit macrophages do not express detectable PKCtheta, PKCtheta mRNA expression was found to be profoundly upregulated during the first hours of lipopolysaccharide (LPS)/interferon-gamma (IFNgamma)-, but not IL-4-mediated cell polarization conditions in vitro. Mechanistically, despite expressing normal levels of classically activated macrophage (CAM) markers, PKCtheta-deficient CAMs expressed significantly higher levels of the anti-inflammatory cytokine IL-10 in vivo and in vitro when challenged with S. typhimurium or LPS/IFNgamma. Neutralization of IL-10 recovered immune control to S. typhimurium infection in PKCtheta-deficient macrophages.

Conclusions

Taken together, our data provide genetic evidence that PKCtheta promotes a potent pro-inflammatory CAM phenotype that is instrumental to mounting protective anti-bacterial immunity. Mechanistically, PKCtheta exerts a host-protective role against S. typhimurium infection, and acts as an essential link between TLR4/IFNgammaR signaling and selective suppression of the anti-inflammatory cytokine IL-10 at the onset of CAM differentiation in the course of a bacterial infection.

     

Anti-θ Serum-indueed Suppression of the Cellular Transfer of Tumour-specific Immunity to a Syngeneic Plasma Cell Tumour

IT has been well documented that tumour-bearing mice can become resistant to their own tumours, especially with chemically induced fibrosarcomas^1–3 and the importance of cell-mediated immune responses rather than humoral antibody in the resistance to tumour transplants has been emphasized^3,4, although the exact mechanism of tumour cell destruction remains ill-defined. Studies in mice^5,6, using allogeneic tumour cells, have demonstrated that thymus-derived (T) lymphocytes are essential for the killing of tumour cells. In addition, using an in vitro method of immunization against histocompatibility antigens, tumour cell destruction either in vitro¹ or in vivo⁸ was shown to be due to T cells alone. In all of these latter studies, however, it is the strong H-2 histocompatibility antigens that are inducing the immune response and not the tumour-specific transplantation antigens (TSTA). We describe here a specific anti-TSTA response to a murine plasma cell tumour which can be transferred with lymphoid cells and which can be shown to involve the essential participation of T cells.     

<Emphasis Type="Italic">Cytisus aeolicus</Emphasis> Guss. ex Lindl. <Emphasis Type="Italic">in vitro</Emphasis> cultures and genistin production

Cytisus aeolicus Guss. ex Lindl. (Fabaceae family, subfamily Faboideae) is an endangered endemic species of the Aeolian Islands, Sicily. In vitro multiplication of C. aeolicus shoots was described in this work and cell cultures were established from cotyledons and hypocotyls to investigate their potential production of isoflavones. Aseptically germinated seeds, cultivated on LS modified basal medium, gave the initial explants used both to induce axillary propagation and callus cultures. The LS (Linsmaier and Skoog) basal medium, supplemented with 0.1 mg L^?1 of 6-benzylaminopurine were used to induce axillary propagation. The callus induction was performed using the basal medium added with 5 mg L^?1 2,4-dichlorophenoxy acetic acid and 5 mg L^?1 kinetin (control medium). Basal medium was also added with 2000 mg L^?1 casein hydrolysate (CH) or 900 mg L^?1myo-inositol (MI). C. aeolicus callus cultures on CH and MI media produced an unique compound, the isoflavone genistein 7-O-ß-D-glucopyranoside (genistin), which has not previously been isolated from wild plants. Callus cultures grown on the medium containing myo-inositol produced the greatest amount of genistin. C. aeolicus tissue culture procedures could provide suitable plant material both for germplasm preservation (by micropropagation) and for biotechnological selective isoflavone production (by callus culture).     

Recombinant flagellin-PAL fusion protein of <Emphasis Type="Italic">Legionella pneumophila</Emphasis> induced cell-mediated and protective immunity against bacteremia in BALB/c mice

We report a new recombinant fusion protein composed of full-length Legionella pneumophila flagellin A and peptidoglycan-associated lipoprotein (PAL), rFLA-PAL, capable of inducing protective immunity against L. pneumophila. The recombinant protein was over expressed in Escherichia coli strain BL21 (DE3) using pET-28a (+) expression vector (pET28a-flaA-pal) and purified by Ni²⁺ exchange chromatography. Immunological properties of rFLA-PAL were assessed in a mouse model. Female BALB/c mice, immunized with rFLA-PAL, exhibited a rapid increase in serum antibody concentration against each of its protein portions. Furthermore, a strong activation of both innate and adaptive cell-mediated immunity was observed as indicated by antigen-specific splenocyte proliferation, IFN-γ and IL-12 production, and early production of TNF-α in the serum and in splenocyte cultures which were separately assessed against PAL and FLA. BALB/c mice were challenged with a lethal dose of L. pneumophila intravenously. In a 10-days follow-up after intravenous lethal challenge with L. pneumophila, a 100% survival rate was observed for mice immunized with rFLA-PAL, same as for those immunized with a sublethal dose of L. pneumophila. Based on the potent immune responses observed in mice immunized with rFLA-PAL, this recombinant fusion protein could be a potential vaccine candidate against the intracellular pathogen L. pneumophila.     

Effect of Anti-immunoglobulin Antisera on Homograft Rejection in Mice

HETEROLOGOUS antisera against immunoglobulins or their component protein chains have been shown to inhibit the immune response in a variety of systems. Antibodies against mouse immunoglobulins, for example, inhibit the response of mouse spleen cells cultured in vitro^1–3. Antibodies against the heavy chains of chicken IgM (anti-μ), administered during embryonation and again at hatching, have produced agamma-globulinaemia in bursectomized chickens⁴, apparently by plasma cell line elimination⁵. Graft-versus-host (GVH) and delayed hypersensitivity reactions have been suppressed in neonatal mice by in vitro pretreatment of injected lymphoid cells with antiserum against light chains⁶. Similar pretreatment with univalent fragments (Fab) of anti-immunoglobulin antibodies has diminished the GVH reaction in adult mice⁷.     

<Emphasis Type="Italic">In vitro</Emphasis> Blastogenesis inhibited by <Emphasis Type="Italic">Erwinia carotovora</Emphasis><Emphasis Type="SmallCaps">L</Emphasis>-Asparaginase

Escherichia coliL-asparaginase, an antileukaemic agent in man¹, inhibits in vitro mitogen or antigen-induced blastogenesis in man^2,3 and in animals (M. Bennett, E. G. Mayhew and T. Han, unpublished data) and suppresses bone-marrow derived antibody precursor cells in the mouse⁴. We now report that another L-asparaginase preparation—from Erwinia carotovora—also possesses antileukaemic activity^5,6 and has a more pronounced immunosuppressive effect on in vitro blastogenesis than the E. coli enzyme.     

Availability of Label in <Superscript>3</Superscript>H-Thymidine for DNA Synthesis in Cultured Macrophages

DURING a study of the developmental potentialities of promacrophages from bone marrow, blood, peritoneal fluid and spleen of rat and rabbit, it was found that a pool of long-lived labelled DNA precursors was formed both in vivo and in vitro while ³H-thymidine was used as label.     

<Emphasis Type="Italic">OsNHX2</Emphasis>, an Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> antiporter gene,can enhance salt tolerance in rice plants through more effective accumulation of toxic Na<Superscript>+</Superscript> in leaf mesophyll and bundle sheath cells

The Na⁺/H⁺ antiporters play an important role in salt tolerance in plants. However, the functions of OsNHXs in rice except OsNHX1 have not been well studied. Using the gain- and loss-of-function strategies, we studied the potential role of OsNHX2 in salt tolerance in rice. Overexpression of OsNHX2 (OsNHX2-OE) in rice showed the significant tolerance to salt stress than wild-type plants and OsNHX2 knockdown transgenic plants (OsNHX2-KD). Under salt treatments of 300-mM NaCl for 5 days, the plant fresh weights, relative water percentages, shoot heights, Na⁺ contents, K⁺ contents, and K⁺/Na⁺ ratios in leaves of OsNHX2-OE transgenic plants were higher than those in wild-type plants, while no differences were detected in roots. K⁺/Na⁺ ratios in rice leaf mesophyll cells and bundle sheath cells were higher in OsNHX2-OE transgenic plants than in wild-type plants and OsNHX2-KD transgenic plants. Our data indicate that OsNHX2 plays an important role in salt stress based on leaf mesophyll cells and bundle sheath cells and can be served in genetically engineering crop plants with enhanced salt tolerance.     

Increase in Salt Tolerance of <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> by <Emphasis Type="Italic">TaDi19</Emphasis>

The gene expression profile chip of salt-resistant wheat mutant RH8706-49 under salt stress was investigated. The overall length of the cDNA sequence of the probe was obtained using electronic cloning and RT-PCR. An unknown gene induced by salt was obtained, cloned, and named TaDi19 (Triticum aestivum drought-induced protein). No related report or research on the protein is available. qPCR analysis showed that gene expression was induced by many stresses, such as salt. Arabidopsis thaliana was genetically transferred using the overexpressing gene, which increased its salt tolerance. After salt stress, the transgenic plant demonstrated better physiological indicators (higher Ca²⁺ and lower Na⁺) than those of the wild-type plant. Results of non-invasive micro-test technology indicate that TaDi19-overexpressing A. thaliana significantly effluxed Na⁺ after salt treatment, whereas the wild-type plant influxed Na⁺. Chelating extracellular Ca²⁺ resulted in insignificant differences in salt tolerance between overexpressing and wild-type A. thaliana. Subcellular localization showed that the gene encoding protein was mainly located in the cell membrane and nucleus. TaDi19 was overexpressed in wild-type A. thaliana, and the transgenic lines were more salt-tolerant than the control A. thaliana. Thus, the wheat gene TaDi19 could increase the salt tolerance of A. thaliana.     

Cryopreservation of <Emphasis Type="Italic">Citrus</Emphasis> anthers in the National Crop Genebank of China

Genebank conservation of pollen is valuable because it makes genetic resources immediately available for use in breeding programs. In the case of Citrus species, conserved anthers or pollen can be easily transported and used to develop new varieties with pathogen resistance and desirable quality and yield traits. The aim of this study was to develop and improve air-desiccation cryopreservation protocols for Citrus cavaleriei and Citrus maxima anthers in genebanks. In the current study, warming, rehydration, and in vitro germination conditions were optimized to achieve high levels of in vitro germination in Citrus pollen for ten cultivars after liquid nitrogen (LN) exposure. The optimal warming, rehydration, and in vitro germination medium formulations affected the germination levels after pollen cryopreservation, with species- and cultivar-dependent effects. The Citrus anthers were dehydrated to the moisture content of 5–14% before LN exposure and warmed at 25 (cryopreserved Citrus anthers with a moisture content of lower than 10%) or 37°C (a moisture content of 10% or higher), then rehydrated, and cultured on medium with 150-g L^?1 sucrose, 0.1-g L^?1 boric acid, 1.0-g L^?1 calcium nitrate, 0.1-g L^?1 potassium nitrate, 0.3-g L^?1 magnesium sulfate, and 10-g L^?1 agar. After 2 yr of storage, in vitro germination levels of Citrus pollen after cryopreservation were significantly higher (> 22% for all ten cultivars) than those of samples that were stored at 4°C (0%). In vitro germination levels of pollen from six of ten cultivars after cryopreservation remained relatively high after 2 yr of storage (38–93%). The highest viability of 93% was obtained for C. cavaleriei ‘2–3’. The methods identified in the current study could be used to cryopreserve C. cavaleriei and C. maxima anthers.     

Stimulation of Autochthonous Lymphocytes by Cells from Normal and Leukaemic Lines

THE mixed lymphocyte reaction (MLR) can possibly be regarded as an in vitro form of an in vivo phenomenon reflecting the recognition of “non-self” tumour specific or neo-antigens on the surface of lymphoid cells. A reaction similar to the normal MLR but of greater magnitude occurs when irradiated lymphoid cells from lymphoblastoid cell lines (LCL) are added to freshly isolated peripheral lymphocytes from allogeneic individuals^1,2. The intense stimulation which occurred in every case when irradiated cells from various LCL were added to lymphocytes from a large number of individuals³ suggested the presence of extra surface determinants on the cells, which are not present on normal freshly isolated cells. We have investigated whether freshly isolated lymphoid cells could detect and respond to extra antigenic determinants on the surface of cell lines derived more than 3 months earlier from their own lymphoid cells.     

<Emphasis Type="Italic">In vitro</Emphasis> Detection of Enhancing Antibody by the Macrophage Migration Test

THE immunological basis of enhanced allograft survival in inbred strains of mice is well established: humoral antibody, by some mechanism which is not yet clear, interferes with the cell-mediated host response to alloantigens¹. Various in vitro correlates of transplantation immunity in the mouse have been developed^2–9 and we have adapted one, the technique of macrophage migration from capillary tubes⁹, as an in vitro method to detect enhancing antibody.