Experimental allogenic transplantation of cornea endothelial cells in cats.

BACKGROUND: The aim of the present study was assessing the possibility of experimental allogenic transplantation of cat cornea endothelial cells, multiplied in vitro, into the anterior chamber of the eyeball in recipient cats. The reason for undertaking the research is the need to develop a method that would help in the cornea treatment in animals with corneal opacification following cataract surgery, as well as lens dislocation, injuries and endothelium degeneration. METHODS: Cats aged 10-12 months were used in the experiment. Cornea fragments consisting of the posterior limiting membrane and posterior epithelium were placed in Iscove's medium with addition of 10% foetal calf serum. Multiplied in vitro cells were injected into the anterior chamber of recipient cats. The cornea was subject to histological, histometric and SEM examination on the 3rd, 7th, 20th and 30th day after the surgery. RESULTS: Micromorphological examination of the cornea showed full restitution of its endothelium 30 days after transplantation. Complete regeneration of structures indispensable for normal functioning of the posterior epithelium occurred as a result of implantation. CONCLUSIONS: In this study the results show that implantation of the cells of posterior corneal epithelium of donor cats, multiplied into vitro and injected into the anterior chamber of recipient cats. The cornea regained its full function, the layer of the posterior epithelium was regenerated and the stroma stabilized, presenting the image of full and proper corneal translucency.

A novel canine B-cell leukaemia cell line. Establishment, characterisation and sensitivity to chemotherapeutics.

We established a new B-cell leukaemia cell line CLB70 from a dog with chronic lymphocytic leukaemia. This cell line is positive for CD20, CD45, CD79a, MHC class II, IgG, IgM; weakly positive for CD21; and negative for CD3, CD4, CD5, CD8, CD14, CD34, CD117. PCR for antigen receptor gene rearrangement (PARR) analysis revealed a biclonal immunoglobulin heavy chain (IgH) gene rearrangement and negative result for TCRγ. Western blot analysis of anti- and pro-apoptotic proteins showed increased expression of Bcl-2, Mcl-1, NF-kB, and Ras, and decreased expression of p53. CLB70 cells grow rapidly in vitro and are tumourigenic in nude mice. The CLB70 line is highly sensitive to doxorubicin, less sensitive to etoposide and imatinib, and resistant to piroxicam, celecoxib and dexamethasone. Our results indicate that CLB70 cells are derived from mature B-cells and they may be a useful tool for the development of new therapeutic strategies for both dogs and humans.

Differential expression of murine leukemia antigen on L1210 parental and drug-resistant sublines.

The different expression of surface antigens on L1210 leukemia DBA/2 and drug-resistant L1210 sublines was investigated. Indirect cytotoxic test, the anti-L1210/v alloantiserum reacted more strongly with subline cells than with parental cells. Absorption of the antiserum with Gross cellular surface antigen-positive AKR leukemia (AKSL-4) cells led to a much greater difference in this reactivity. Quantitative absorption experiments revealed that the drug-resistant sublines had 5 times higher absorption capacity than did the parental line. After complete absorption of antibodies against murine leukemia virus-related antigens, the anti-L1210/v alloantiserum still reacted with L1210 cells. This cytotoxicity could be removed after absorption with C3H mammary tumor (MAC-1) cells but not with normal C3H lymphocytes. These results provide evidence that the major cytotoxic activity of the antiserum against L1210 and L1210 subline cells was due to antibodies against murine mammary tumor virus-related antigen and that the drug-resistant sublines of leukemia L1210 have higher quantitative expression of mammary leukemia antigens.

Cytotoxic interactions of bare and coated NaGdF4:Yb(3+):Er(3+) nanoparticles with macrophage and fibroblast cells.

The lanthanide nano-compounds are well suited to serve as fluorescent and magnetic contrast agents and luminescent labels. Although they are considered as promising materials for bio-imaging and bio-sensors in vivo or in vitro, the amount of data is still insufficient for deep understanding the toxicity of these nanomaterials. This knowledge is of great importance in the light of growing use of the biofunctionalized nanoparticles, which raises some questions about safety of these materials. Despite lanthanide-doped NaGdF4 nanocrystals are considered as non-toxic, here we present the data showing the fatal effect of newly synthetized NaGdF4:Yb(3+):Er(3+) on chosen types of cells. Our studies were performed on two cell lines NIH3T3 fibroblasts, and RAW264.7 macrophages. Cytotoxic properties of NaGdF4:Yb(3+):Er(3+) nanoparticles and their biological effects were studied by assessing cell culture viability (MTS), proliferation and apoptosis. Bare NaGdF4:Yb(3+):Er(3+) nanocrystals were cytotoxic and induced apoptosis of both NIH3T3 and RAW264.7 cells. Their cytotoxicity was reduced by PEGylation, at the expense of minimizing direct interactions between the compound and the cell. On the other hand, coating with silica reduced cell death induced by Yb(3+):Er(3+) codoped NaGdF4 nanocrystals (but proliferation was still inhibited). The NH2-modified silica coated nanoparticles were clearly less cytotoxic than pristine nanoparticles, which suggests that both, silica and PEG coatings are reasonable approaches to decrease cytotoxicity of the nanocrystal labels. The silica and PEG shell, should also enable and simplify further bio-functionalization of these luminescent labels. The authors acknowledge the financial support from: Institute of Immunology and Experimental Therapy, Polish Academy of Sciences (IITD PAN) grant no. 3/15, Polish Ministry of Science and Higher Education, Grant N N507 499538 and from the Wroclaw Research Centre EIT+ within the project "The Application of Nanotechnology in Advanced Materials" - NanoMat (POIG.01.01.02-02-002/08) financed by the European Regional Development Fund (Operational Program Innovative Economy, 1.1.2).

Lectin-resistant variants of mouse Lewis lung carcinoma cells. I. Selection and in vivo properties.

The availability of lectin-resistant cell lines with altered carbohydrate moieties in cell surface glycoproteins and glycolipids has greatly facilitated study of the involvement of cellular glycoconjugates in tumor growth and metastasis. We present here a new animal model for metastasis study based on mouse Lewis lung carcinoma LL2 in vitro cell line. From this line, five lectin-resistant variant sublines were selected with the following lectins: wheat germ agglutinin (WGAR), Ricinus communis agglutinin II (RCA IIR) and Aleuria aurantia agglutinin (AAAR). The correlation of the lectin resistance with their in vitro and in vivo growth properties, and especially lung colonizing ability, were investigated. Three WGAR variants with well-preserved tumorigenicity revealed reduced metastatic ability, both spontaneous, after subcutaneous (s.c.) administration and experimental, after intravenous (i.v.) administration. The RCA IIR variant also possessed reduced spontaneous and experimental metastatic ability, but exhibited higher growth rate of local s.c. tumors. The AAAR variant possessed reduced spontaneous metastatic ability but its ability to colonize the lungs after i.v. administration was five-fold higher than that of the parent LL2 line, whereas its tumorigenicity remained unchanged. The relative differences among WGAR variants and parent LL2 line, concerning their experimental metastatic ability, remained similar in cyclophosphamide-modified mice to those in normal recipients.

Overexpression of Ras, Raf and L-myc but not Bcl-2 family proteins is linked with resistance to TCR-mediated apoptosis and tumorigenesis in thymic lymphomas from TCR transgenic mice.

Mice with transgenic TCR anti H-Y/Db develop spontaneous thymic tumors with a high frequency (up to 50%). Oncogenicity of TCR transgenes could depend on the deregulated expression of oncoproteins engaged in transduction pathways leading to proliferation or apoptosis. In agreement with this possibility we have found that cells of thymic lymphomas from TCR transgenic mice were largely resistant to TCR-dependent Ca++-mediated apoptosis but not to TCR-independent, p53-mediated (etoposide) apoptosis. Here we show raised expression of Bcl-2 protein in some but not in all thymic lymphoma cell lines. It suggests that the antiapoptotic function of Bcl-2 is not necessary for the process of tumorigenesis and the resistance of these lymphomas to Ca++-mediated apoptosis. On the other hand we show that all thymic lymphomas overexpressed Ras/Raf and L-myc proteins. Stimulation of the Ras/Raf pathway was reported to be required to maintain cell viability by preventing programmed cell death in thymic tumors derived from lck transgenic mice. Similarly, in TCR transgenic lymphomas overexpression of Ras, Raf and L-myc but not Bcl-2 family proteins may be responsible for the resistance of these lymphomas to TCR-mediated apoptosis but not affect p53-mediated apoptosis.

Enhancement by cyclophosphamide of experimental pulmonary metastases formation of Lewis lung carcinoma. I. Dose-dependence and kinetics of cyclophosphamide effect.

The results presented in this study indicate that treatment of mice with Cyclophosphamide prior to Lewis lung carcinoma (LL2) cells inoculation enhanced the formation of artificial lung metastases in a dose-dependent manner. At the dose of Cyclophosphamide equal to 200 mg/kg the number of lung colonies was increased by a factor varying between 8 and 32. The effect was also time-dependent and it persists up to seventh day after Cyclophosphamide administration but was not visible when LL2 cells were injected 2 or 3 weeks later. Treatment of mice with Cyclophosphamide after LL2 cells inoculation did not enhance formation of tumor metastases.

Enhancement by cyclophosphamide of experimental pulmonary metastases formation of Lewis lung carcinoma. II. Reduction of the cyclophosphamide effect by repopulation with cells from lymphoid organs.

Cyclophosphamide-induced enhancement of artificial lung metastasis of LL2 could be partly abolished by reconstituting of mice with spleen, lymph nodes or bone marrow cells but not with thymus cells. At cell dose of 10 x 10(6) per mouse the highest reconstituting capacity was found for spleen cells (73%) followed by bone marrow (60%) and lymph nodes cells (22%). Cell dose of 10(6) lymphoid cells per mouse did not reduce the CY-effect. The higher cell number (30 x 10(6) and 50 x 10(6) per mouse) did not abolish completely the CY-effect. The results indicate that in addition to sensitive lymphoid cells there exists another target for CY effect.

Interleukin 3-dependent, asialo-GM1-positive cell line with natural cytotoxic but without natural killer cytotoxic activity.

IL-3-dependent cell line (designated as A-3) from adherent spleen cells was developed. The surface phenotype of A-3 cells was analysed by flow cytometry and determined as ASGM1+, FcR+, Ia+. There was no expression of T cell markers (Th1-CD3-CD4-CD8-), B cell marker (surface Ig) as well as macrophage marker Mac-1 antigen. Although A-3 cells expressed ASGM1, they are lacking NK cytotoxic activity in 4 h Cr release assay with YAC-1 targets. In contrast, A-3 cells are endowed with NC cytotoxic activity as shown in 18 h Cr release assay with WEHI-164 target cells.

Studies on cell surface antigens of mouse leukemic and normal lymphocytes. II. Relationships between mammary leukemia, gross virus related and major histocompatibility antigens on L1210 leukemic lymphocytes.

The presence of ML antigen on leukemia L1210 cells was confirmed by different procedures. In addition, by applying appropriate absorptions and blocking techniques combined with ferritin labelled antibodies in electron microscopy (EM), the independent localization of ML antigen and murine leukemia virus Gross (MuLV-Gross) cellular antigen on L1210 cells was demonstrated. The ferritin labeled antigenic sites appeared to be free from budding viral particles. In further studies the 3M KCI extracts of L1210 leukemia cells purified by polyacrylamide gel electrophoresis (PAGE) and the antigenic material from leukemia L1210 cells purified by affinity chromatography were characterized. Their identity was verified by anti ML-sera. Using the modified lyso-strip method, the presence of ML antigen and H-2K and H-2D gene products on independent molecules on the surface of L1210 cells was shown. In addition, in preparative polyacrylamide gel electrophoresis (PAGE) of 3M KCL extracts of L1210 cells, ML and H-2d antigenic products were found in separate fractions.

Expression of Thy1, TL and LYT antigens on spontaneous leukemias of BALB/MO strain of mice.

The expression of Thy1, TL and Tyt antigens on spontaneous BALB/Mo leukemias has been studied. The results obtained thus far indicate that a majority of leukemias express TL antigen and differentiated Lyt phenotype. This observation and other findings demonstrating the existence of TL+ thymocytes with different Lyt phenotypes in normal thymus of young mice lends support for the hypothesis that TL+Lyt2+ phenotype defines this stage in the T cell differentiation pathway, which is particularly sensitive to leukemic transformation.

Transplantable mouse 16/c mammary adenocarcinoma as a model in experimental cancer therapy. II. Modification of tumor-host interactions by therapeutic procedures.

The influence of therapeutic procedures on tumor--host interactions was analysed using 16/c mouse mammary adenocarcinoma model system. After partial removal of tumor burden the growth enhancement of remnant tumor mass was observed. Pretreatment of the host either with cyclophosphmide or X-rays resulted in enhancement of experimental metastasis formation. Unexpectedly, such a procedure caused the growth delay of the primary subcutaneous tumor. The possible mechanisms of observed phenomena are discussed.

Transplantable mouse 16/C mammary adenocarcinoma as a model in experimental cancer therapy. I. Kinetics of growth and spread.

Introduction of transplantable animal tumours has enabled the development of different model systems in which the site and type of tumour growth depends on the selection of suitable route for cells inoculation. Manipulation of the inoculation route of 16/C mammary adenocarcinoma results in local tumour growth either subcutaneous (s.c.), intramuscular (i.m.), intraperitoneal (i.p.) or intrasplenical (i.spl.) and development of metastases in lungs, lymph nodes, and liver. Exponential model of s.c. growth of the tumour was estimated and doubling time value calculated (3, 64 days). Correlation between the presence of tumour cells in lungs and advancement of s.c. growing tumours was found. Usefulness of 16/C adenocarcinoma as a model for experimental therapy is discussed.

High expression of endogenous bcl-2 and bcl-xL in thymic lymphomas do not diminish their sensitivity to etoposide-induced apoptosis.

It was previously reported that thymic lymphomas of anti HY-TCR transgenic mice were resistant to ionomycin but were sensitive to 50 microM of etoposide. We selected lymphoma clones with strong expression of anti-apoptotic proteins Bcl-2 or Bcl-xL and found that in contrast to proteins encoded by exogenous genes in other model systems, over-expression of endogenous Bcl-2 and Bcl-xL proteins failed to protect against etoposide-induced apoptosis. Susceptibility of lymphoma cells was not diminished even to suboptimal concentration of 0.5 microM of etoposide. Treatment with etoposide did not decrease the ratio of anti-apoptotic Bcl-2 and Bcl-xL proteins to pro-apoptotic Bax protein. Results presented here suggest that high expression of Bcl-2 and Bcl-xL may not diminish susceptibility of T-cell lymphomas to chemotherapy with etoposide.

Unexpected reaction of anti-H-2d serum with thymic lymphoma cells derived from transgenic B6-H-2b TCR anti-HY/Db mice.

Transgenic mice with alpha beta TCR specific for male antigen (HY) in the context of H-2Db MHC class I were prepared by introduction of transgens into (C57BL/6J x DBA/2J)F1 hybrid mice (H-2b/d), where only H-2b is selective for maturating thymocytes. Founder transgenic mouse was backcrossed to H-2b MHC background. Serological typing of H-2 antigens revealed that transgenic mice do not express H-2d antigens. Unexpectedly, cells of thymic lymphomas, spontaneously developed in about 45% of old B6 (H-2b) transgenic mice and peripheral blood lymphocytes of about 40% of transgenic mice, reacted with anti-H-2d serum (strain 129 mice (H-2b) immunized with thymocytes and splenocytes of BALB/c mice) but not with monoclonal antibodies against H-2d MHC class I antigens. Anti-H-2d serum has been shown to react with thymocytes but not peripheral blood lymphocytes from non-transgenic H-2b mice. The lymphocytes of transgenic mice reacting with anti-H-2d serum could represent disseminating preneoplastic or neoplastic cells expressing antigen encoded outside MHC region and present on the cell surface of immature thymocytes but not lymphocytes in healthy mice.

Loss of T cell receptor diminished tumorigenicity of thymocyte-derived lymphoma cells in the T cell receptor transgenic mice.

Mice with transgenic T cell receptor (TCR) recognizing H-Y male antigen developed spontaneous lymphomas originated from immature thymocytes, with the surface expression of transgenic TCR and CD4/CD8 co-receptors. During in vitro long-term culture (3 months) some lymphoma cell lines lost the surface expression of TCR and co-receptors. Interestingly, the proteins of transgenic receptor were expressed intracellularly but TCR was not detectable on the surface of the in vitro selected subline in contrast to TCR-positive parental cells maintained in vivo. TCR-negative subline has been found to be slowly growing in vivo (i.p. injection) and less tumorigenic (s.c. injection) than parental TCR positive lymphoma. It seems that the in vivo interactions of lymphoma cells with microenvironment preserve their TCR expression and endow with growth advantage, while the selected in vitro TCR-negative cells lose the tumorigenic potential.

Changes in glucocorticoid-induced apoptosis and in expression of Bcl-2 protein during long-term culture of thymic lymphoma.

Lymphomagenesis is a multistep process progressively freeing transformed thymocytes from external regulatory signals, i.e. thymic developmental program controlling growth, differentiation or apoptosis. Here we report that cells of thymic lymphoma overexpressing Ras/Raf proteins, initially resistant to TCR-dependent apoptosis but sensitive to dexamethasone- and etoposide-induced cell death, became insensitive to dexamethasone after long-time culture. That transition correlated with a strong increase in the expression of the anti-apoptotic Bcl-2 protein. Interestingly, lymphoma cells were still sensitive to p53-mediated apoptosis induced by etoposide. It suggests that the anti-apoptotic activity of Bcl-2 is correlated with a resistance to glucocorticoid-induced apoptosis but not to p53-mediated apoptosis. The sequence of mutations in the process of lymphomagenesis seems to be composed of at least 3 main hits which equip the cells with independence from external mitogenic signals (activation of Ras/Raf), resistance to inducers of apoptosis (activation of Bcl-2) and generation of cellular heterogeneity (deletion of p53) important in tumor progression.

Molecular size and amino acid composition of H-2d antigen solubilized in Nonidet P-40.

H-2d antigenic material solubilized by the detergent Nonidet P-40 from L-1210 mouse leukemia cells was isolated by gel filtration on Bio-Gel P-100. A single peak eluted in the void volume consisted of about 90% protein, 8% hexose and traces of sialic acids. In sedimentation velocity runs, the antigen sedimented as a single peak of 3-1 S. Molecular weight determined by sedimentation equilibrium as well as calculated from amino acid composition was found to be in the range of 53,000 daltons and approx. 45,000-51,000 when calculated from sodium dodecyl sulfate polyacrylamide gel electrophoresis. Secondary structure of H-2d glycoprotein was predicted from the amino acid composition. For NP-40-solubilized H-2d antigen, about 34% of helix, 13% beta sheet and 41% turns was found.

Transplantable mouse mammary adenocarcinoma 16/c as a model in experimental cancer therapy. III. Sensitivity to antitumor drugs.

On the basis of biological characteristics of murine transplantable mammary adenocarcinoma 16/c, experimental conditions optimal for chemotherapeutic experiments were defined. Sensitivity of primary tumor and lung metastases to the treatment with drugs used in breast cancer therapy: cyclophosphamide, 5-fluorouracil and adriamycin was confirmed. These drugs were significantly more effective in combination than when administered as monotherapy. Antimetastatic but not antitumor effect of 5-fluorouracil could be potentiated by using albumin microspheres as a carrier. Usefulness of the tumor model system in experimental chemotherapy is discussed.

Potentiation of cyclophosphamide-induced enhancement of experimental metastasis by splenectomy.

Role of spleen in CY-induced enhancement of experimental lung metastases of Lewis Lung Carcinoma LL2 cells was studied. Reconstitution with spleen cells abolished the enhancing effect of CY. Conversely, removal of spleen in CY treated mice caused about two-fold increase in the number of metastases. In addition in splenectomized mice, the effect of CY was augmented.