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The role of MRP4 and MRP5 transporters in the acyclic nucleoside phosphonate PMEDAP efflux was studied in vitro (CCRF-CEM cells) and in vivo (spontaneous transplantable T-cell lymphoma of SD/Cub inbred rats). The increased resistance against the cytostatic agent PMEDAP during longterm treatment was found to be associated with overexpression of MRP4 and MRP5 genes. The course of both gene activation differs significantly. While the MRP5 function is important in the onset of PMEDAP resistance, the intensity of the relative MRP4 gene expression increases rather continuously. Our data indicate cooperative acting of both MRP4 and MRP5 genes during the PMEDAP resistance development.

More than a decade of experimental work in an inbred subline of Sprague-Dawley rats having high incidence of spontaneous T-cell lymphoma/leukaemia is reviewed. Longitudinal follow-up of biological characteristics (growth, survival, haematology) of both multiple cases of primary disease and s.c. passaged lymphomas as well as comparative immunophenotypic and karyotypic studies are concluded. In these T-cell lymphomas (mostly CD4 positive), arising on the same genetic background of the inbred SD strain, the aberrations involving chromosome 11 have been recognized as a typical non-random cytogenetic marker. This unique rat model of lymphoblastic lymphomas/leukaemias, relevant to human pathology, seems to be very suitable for testing different anticancer therapeutic strategies, as it is documented by results of a number of various protocols conducted in our laboratory.

Anticancer effect of heat shock, either alone or in combination with the drug PMEDAP, and cold water immersion stress were studied in an in vivo model of s.c. transplanted rat T-cell lymphomas in an inbred Sprague-Dawley rat line (SD/cub). Significant anticancer effect was induced by repeated sessions of heat shock; decrease of s.c. lymphoma weight and prolongation of survival time of treated rats was found to be dependent on the number of HS sessions. Much stronger therapeutic effect was observed after repeated heat shock in combination with PMEDAP administration. Light and electron microscopy studies were performed to characterize the alterations within the lymphomas. Morphologically, cellular alterations corresponding with apoptosis were observed in lymphoma cells after repeated heat shock. Indirect immunoperoxidase technique was used to detect HSP 72/73 protein(s), p53 and Bcl2 proteins in lymphomas heated directly or indirectly. The induction of HSP 72/73 protein(s) was found in the lymphoma tissues from autopsied animals exposed to heat shock; the intensity of its expression was dependent on the experimental design. The expression of p53 and BcL2 proteins was not changed in lymphoma cells of HS treated animals as compared to that of untreated lymphoma bearing controls; the Bcl2 protein was present in both treated and untreated lymphomas, and the p53 protein remained undetectable in all samples. Contrary to the heat shock, the cold stress did not suppress growth of lymphomas and, furthermore, accelerated the infiltration of parenchymatous organs with lymphoma cells.

Changes in the karyotype in three spontaneous Sprague-Dawley rat lymphomas (SD7/95, SD8/96, SD9/96) have been studied in the course of in vivo passages. In individual lymphomas karyological findings of primary disease from lymph nodes were compared with changes found in the 1st and 10th passages of the lymphoma and bone marrow samples. Chromosome studies were performed on direct preparations using the G-banding technique. Chromosome counts of all specimens studied were near diploidy, the majority of metaphase cells being pseudodiploid. In later passages of two lymphomas, the tendency in selection to hyperdiploid karyotype, particularly in bone marrow was observed. The examination revealed an increased percentage of breaks in lymph node cells of primary disease and the existence of nonrandom change, derivative chromosome 11, which occurred in structural variability in all three lymphomas studied. The aberration involving chromosome 11 was evaluated as the addition of unknown material at chromosome band 11q11 or as a duplication or triplication of segment 11q12-q23. If this structural aberration was not found, the excessive derivative chromosome 11 or translocation t(11;13) was proved to be present. Further, rearrangements of chromosomes 13 and 7 were nonrandom chromosome abnormalities revealed in later passages of the lymphomas. The results are in accordance with our previous observations in 14 cases of SD lymphomas that showed nonrandom occurrence of rearrangements concerning chromosome 11 and also relatively frequent translocation involving chromosome 13.

Spontaneous rat CD4+CD8-T-cell leukaemia transplanted in syngeneic recipients served as an experimental model system for IL-2 therapy. As a source of IL-2, supernatants from in vitro cultured plasmacytoma cell line X63-m-IL2 secreting constitutively recombinant murine IL-2 were utilized. Administration of IL-2, s.c. to the vicinity of the tumour inoculum, suppressed tumour growth. The tumour-inhibitory IL-2 effects were time- and dose-dependent. When the treatment has started 10 days after the challenge with 10(4) leukaemia cells, IL-2 inhibitory effects on the lymphoma growth in situ were demonstrated by lower tumour weight combined with necrotic changes. No histological signs of lymphoma generalization were found in parenchymatous organs of IL-2-treated rats in contrast to the untreated controls. No histological or functional injuries to the kidneys due to IL-2 administration were found. The results of effector cell phenotyping demonstrated the kinetics of the CD4+/CD8+ ratio characterized by CD4+ T-cell depletion and resulting increase in the percentage of CD8+ PBL.

The genotoxic and embryotoxic effects of phosphonomethoxyalkylpurines, a new group of antiviral agents, decrease in the following order: PMEG > PMEthioG > PMEDAP > PMEA > (R)-PMPDAP = (R)-PMPA. Results of the present study are fully consistent with the previously found efficacy of their diphosphates to inhibit the replicative DNA polymerases. The marked genotoxicity of PMEG and PMEthioG is comparable to that of mitomycin C, whereas the moderate genotoxicity of PMEA is comparable to that of AZT. (R)-PMPDAP and (R)-PMPA did not induce any structural aberrations of chromosomes under the experimental conditions.

The antiphlogistic Ibuprofen incorporated in liposomes caused a decrease of the inflammatory edema induced by Carrageenan in the distal part of the rat's hind leg after both the intramuscular and percutaneous administration. The antiphlogistic effect of free Ibuprofen in the cream was weaker. Intramuscular administration of empty liposomes slowed down in the initial stages the development of inflammation and slightly diminished the size of edema.

To verify the penetration of liposomes through skin, we have used liposomes with encapsulated protein G-gold conjugate in a gel vehicle. Skin samples were examined 2, 4, 24, 48, and 72 h after liposome application. Our findings show that the penetration of liposomes through skin depends mainly on their size. Liposomes up to 600 nm in diameter penetrate through skin rather easily, whereas liposomes 1000 nm and more in diameter remain interiorized in the stratum corneum. The main penetration of liposomes proceeds along the hair sheaths as indicated by larger amounts of free liposomes in the corium of guinea pigs.

We have investigated the interaction of liposomes with the continuous cell lines P388D1 and L-132 and mouse peritoneal macrophages. To distinguish the liposomes from other vesicular structures, we have used liposomes with incorporated protein G and gold. A heterogeneous mixture of multilamellar liposomes 30 nm up to 1000 nm in size has been employed. Samples were examined at different time intervals. We found differences in the uptake of liposomes with regard to size and rate. Cells of a P388D1 monolayer took up liposomes by endocytosis very early after addition of liposomes and the number of lysosomes in their cytoplasm increased. In L-132 cells, first a fusion occurred between liposomes and the cell cytoplasmic protrusions, in the cytoplasm of which the mitochondria had multiplied. Peritoneal macrophages phagocytosed mainly large multilamellar liposomes and the membranous system of Golgi apparatus was the most prominent structure in the cytoplasm. Phagocytosis in P388D1 and L-132 cells was noted sporadically as late as 24 h after addition of liposomes to the cells.

PMEA was found to influence the immune system of the mouse and rat in vivo in different ways. PMEA administered after injection of parental splenocytes to F1 recipients reduced the development of local graft-versus-host reaction (GVHR). However, PMEA treatment of donors of splenocytes had no influence on GVHR. Modifications in the immune system triggered by PMEA were confirmed in the rat model by evaluation of subsets of white blood cells isolated from peripheral blood by a set of monoclonal antibodies. Enhanced formation of nitric oxide (NO) was found in both unconditioned and LPS-stimulated macrophage cultures on day 14 of the drug treatment of rats.

Retrovirus-like particles were detected by the negative staining method in supernatants of lymph node and spleen cell suspensions prepared from Sprague-Dawley rats with spontaneous acute lymphoblastic leukaemia (ALL). Similar particles were found in supernatants of cell suspensions from a lymphoma that developed after inoculation of lymph node and spleen cell suspension prepared from animals with spontaneous ALL into the subcutis of Sprague-Dawley recipients. On ultrathin sections, budding forms of the virus particles were seen as a dot at the periphery of neoplastically transformed cells.

Five cases of spontaneous acute lymphoblastic leukaemias (ALL) of Sprague-Dawley (SD) inbred rats have been studied cytogenetically. Chromosome studies were performed on direct preparations of the lymphomas using common cytogenetic methods. For detailed karyology, G-banded preparations were used. The chromosome numbers of metaphase cells in all leukaemias were near diploidy, the majority of metaphases being pseudodiploid with 1-2 stable marker chromosomes. The most frequent change involved chromosome 11, where the translocation form of trisomy 11 or 11q+ aberration was observed. Chromosome 11 abnormalities were found in all five leukaemias studied, suggesting to be a nonrandom change. 11 q11-q12 is supposed to be the critical region involved in the genesis or progression of SD ALL. These results are in agreement with our previous observations in nine cases of SD ALL (Sladká et al., 1988).

Two cases of spontaneous acute lymphoblastic leukemia in the inbred strain of Sprague-Dawley (Prague) rats have been observed. Since its reliable transplantability in syngeneic recipients was established, leukemic animals were used to test the cytostatic effect of PMEA. The treatment resulted in a significant prolongation of survival time of the treated animals. At the same time histological examination of PMEA-treated and untreated animals indicated that the drug effectively slows down the growth of lymphoma at the site of inoculation and inhibits the subsequent progression of tumor cells in the lung, liver, spleen and lymph nodes.

Severe graft-versus-host disease (GVHD) developed following intravenous administration of parental BN.lx lymphoid cells into (SHR x BN.lx)F1 animals. Clinical symptoms, including foot-pad hyperemia, slobbering, melaena, progressive weight loss leading to death within 3 to 4 weeks, can be completely abrogated by 8 injections of PMEA starting 1 h after cell administration. Practically normal histological findings in PMEA-treated animals contrasted with nearly complete damage of bone marrow, lymphoid infiltration of salivary glands and even ulceration with hemorrhage of the epidermoid part of the stomach in untreated control GVHD animals.

PMEA was administered i.p. daily on 10 consecutive days to inbred LEW rats inoculated with leukemic lymphoblastic cells KPH-Lw-I. The treatment was started 24 h after the inoculation. The observed cytostatic effects consisted in a significant prolongation of survival time associated with a suppression of the number of bone marrow leukemia cells with characteristic chromosomal marker of KPH-Lw-I leukemia as well as with a depression of the number of lymphoblasts in the blood.

The cytostatic potency of PMEA was evaluated on five rat and mouse experimental tumors. The compound significantly inhibits three spontaneously arisen (LW13K2, A870N and LLC) tumors. Two chemically induced tumors (FBN, HEP-LEW) were not influenced by PMEA.

The acyclic nucleotide analogues, HPMPC and PMEA, differ in their in vitro effect on the genetic material of eukaryotic cells. While HPMPC exerts a cytostatic effect on eukaryotic cells in vitro, PMEA has a genotoxic activity. These results correspond with the mode of embryotoxic action of these compounds: HPMPC exhibits a general embryolethal effect, whereas PMEA is apparently teratogenic and interacts with the mutant allele producing preaxial polydactyly of the hind limbs.

MRC OX-3, MRC OX-6, and MRC OX-17 antibodies directed against rat MHC class II antigens were tested on a panel of rat inbred and congenic strains with different haplotypes using indirect immunoperoxidase and immunofluorescence techniques. In accordance with reports of others the MRC OX-17 was found to define RT1 class II monomorphic and the MRC OX-3 polymorphic determinants. MRC OX-6, giving no reaction with RT1d and RT1dv1 haplotypes, was shown to be directed against a rat MHC class II polymorphic determinant.

From a panel of seven mouse monoclonal antibodies against human class II molecules only one, HL-37, directed against the beta chain of human DQ1 and DQ3 antigens cross-reacted in membrane immunofluorescence with the RT1b haplotype of the rat major histocompatibility complex. From the results obtained using an indirect immunoprecipitation method with 35S- and 125I-labelled rat samples from RT1b-carrying lymph node cells it can be supposed that the rat homologue of the human DQ beta chain was detected.

In rats, two alloantigenic loci, RT4 and RT6, were separated by recombination in a backcross population of (LEW X LEW.C-4A) X LEW hybrids using cytotoxicity tests with alloantisera and immunofluorescence with monoclonal antibody P4/16 in combination with histogenetic (skin graft) testing. Two new recombinant congenic strains were established, LEW.C-4A/I of the genotype CCRT4aaRT6aa and LEW.6B of the genotype ccRT4bbRT6bb. In the rat, transplantation results localize the histocompatibility locus RT4 closer to the locus albino, the T-cell RT6 locus without any proven histocompatibility effect is closely linked to the haemoglobin locus Hbb, in contrast to the mouse in which the haemoglobin (Hbb) and histocompatibility H-1 loci are closely linked.