[Thromboelastography in liver transplantation, a comparison with conventional laboratory tests]. / Trombelastograf pri transplantaci jater, porovnání s konvencními koagulacními testy.

BACKGROUND: The aim of our study was to compare the results of conventional tests and thromboelastography during liver transplantation and to determine their importance for blood loss. METHODS AND RESULTS: Thromboelastography and conventional laboratory tests were undertaken in 25 patients at the end of the anhepatic phase. Transfusion requirements correlated significantly only with prothrombin time and reaction time, R. These two tests likewise correlated significantly one with the other. CONCLUSIONS: Lowered plasma levels of coagulation factors of the prothrombin complex influenced the blood losses in our patients. While not replacing conventional tests, thromboelastography can serve as an additional test for monitoring acute changes in hemostasis.

Heart targeting of retroviral expression in avian embryos: a species-independent phenomenon.

A replication-incompetent retroviral vector derived from spleen necrosis virus (SNV), in which the viral structural genes gag, pol, and env were replaced with the bacterial ß-galactosidase gene lacZ, was used to infect embryos from outbred and inbred chicken lines, japanese quail and duck between embryonic day 0 and 13. LacZ expression was restricted to a few organs or cell types, and this distribution was not influenced by the different routes of inoculation tested but was specified by the age of the embryo at the time of inoculation. Inoculations at E0-E1 beneath or onto the blastodisc resulted in lacZ expression in ectodermal derivatives, i.e. skin and neural structures. From E2 onwards, heart muscle and skin were the preferential targets in all the species or inbred lines tested. Heart muscle was positive in 100% of the embryos displaying lacZ+ clones. Skin exhibited on and off periods depending on the age at inoculation. No lacZ-positive clones were detected in chick embryos infected after Ell. Outbred chick embryos displayed the largest array of organs labelled (heart, skin, liver, gizzard) while quail and duck embryos exhibited a more restrictive pattern. These results are of import if the vector is to be used as a tool to map lineages or to transfer genes into the developing embryo.

The major histocompatibility complex of chickens controls the infection of early chicken embryos by MC29 virus.

Embryos from isogeneic chicken lines belonging to different haplotypes and known to be resistant to infection by avian retroviruses of subgroups A and E were infected on the 3rd (E3) and 5th day (E5) of incubation with MC29 virus (MC29-RAV-1 pseudotype; A subgroup-derived envelope). Despite the trait for resistance, E3 embryos developed the specific heart tumors previously described in outbred E3 embryos. The CB line (B12/B12, C/AE) was more susceptible than the congenic line CC (B4/B4, C/AE). In both lines, the heart was the unique target at E3 for MC29. No tumors of the heart or other organs appeared upon infection at E5 or E10. In the A subgroup susceptible line 6 (B2/B2, C/E) both heart (50%) and skin (100%) were transformed upon E3 infection. Hybrids of line 6 with the CB line expressed skin (100%) and heart (95.4%) tumors. On the other hand, the 6 x CC combination revealed 96.7% of skin tumors while heart tumors occurred only in 1 of 31 embryos (3.2%). To distinguish the respective influences of the MHC and of the tv-a allele, crosses with the la line (B7/B7, C/O) were carried out and tested with MC29. The findings indicate that resistance of the embryos to MC29 heart tumors is associated with the B4/B4 haplotype, supporting the interpretation that the MHC has a role in MC29 cell tropism and v-myc expression. The target cells in tumors were determined by immunofluorescence staining. Cells infected in the heart belonged to the myogenic lineage, as expected from previous studies. In skin anomalies the epidermal cells were double-stained with anticytokeratin and anti-env antibodies, many cells in the dermis also reacted with anti env antibodies.

c-myc gene activation as a permanent trait of RSV-infected quail cells.

A high level of c-myc gene expression was found to be a constant feature of v-src transformation. The c-myc gene product was analyzed in quail embryo cells transformed by different mutants of Rous sarcoma virus (RSV) that were temperature-sensitive with respect to various parameters of v-src function. The high-level expression of c-myc proved not to be temperature-sensitive: at both permissive (35 degrees C) and non-permissive (41 degrees C) temperatures, the same high levels of c-myc gene product were found for all RSV mutants tested. This result, in agreement with earlier evidence for a v-src-induced proliferative stimulus which was unaffected by ts mutants at the non-permissive temperature, shows that certain v-src functions have not yet been fully characterized.

Role of replaced v-src and env genes in the duck-adapted variant of Rous sarcoma virus.

The env and v-src genes of a duck-adapted variant of Rous sarcoma virus were replaced for corresponding genes from parental chick-derived virus. The generation of viral constructs with replaced genes is described. DNAs of viruses with replaced genes were assayed on chick and duck embryo fibroblasts by transfection assays. Transformation efficiency was measured by the focus assay and multiplication of virus by the reverse transcriptase assay. Duck-adapted virus with replaced env gene lost the higher transformation efficiency for duck cells, whereas replacement of the v-src gene had no effect on its host-specific transformation activity.

Inhibition of hepatitis B virus surface gene expression by antisense oligodeoxynucleotides in a human hepatoma cell line.

We have studied the inhibitory effect of antisense oligodeoxynucleotides on the expression of hepatitis B virus surface antigens. Human hepatoma cell line PLC/PRF/5 harbors several integrated copies of the HBV genome and produces and secretes hepatitis B virus surface antigen (HBsAg) to the medium. Synthetic antisense oligodeoxynucleotides complementary to various regions of the surface antigen gene were synthesized and their ability to block its expression was tested. Oligodeoxynucleotides (17- and 21-mers) complementary to regions covering ATG codons of both preS2 and S genes significantly inhibited preS2 and S protein production. Less efficient inhibition was achieved when the oligonucleotide complementary to the inside S gene region was assayed.

Biology of Rous sarcoma virus adapted for replication and transformation of duck cells.

A duck-adapted variant of the Prague strain of Rous sarcoma virus and its molecularly cloned derivatives were characterized biologically. The virus replicated and transformed with the same efficiency both duck and chicken embryo fibroblasts. The daPR-RSV-C was also used for induction of tumours in newborn hamsters. The virus from virogenic hamster tumours could be rescued using the duck cells as indicator cells. The virus rescue was influenced by restricted replication of the virus in the indicator cells.

Monoclonal antibodies against hepatitis B e antigen: production, characterization, and use for diagnosis.

Five different hybridoma clones secreting anti-HBeAg antibody were constructed by fusing cells of mouse myeloma line SP2/0 with splenocytes from BALB/c mice immunized with recombinant HBeAg. The monoclonal antibodies obtained were characterized immunologically and one was used to develop ELISA for detection of HBeAg and anti-HBeAg antibody. These monoclonal assays enabled the detection of 3 U HBeAg/ml and 1 U anti-HBeAg/ml with reference to standards of the Paul Ehrlich Institute, Frankfurt, F.R.G. Both assays compared well with a commercially available kit (Abbott Laboratory) and were used for detection of HBeAg and anti-HBeAg antibody in clinical serum samples.

Avian bone-derived cell line infected with osteopetrosis virus pts-56.

Cell culture derived from embryonal turkey bone was infected with osteopetrosis virus pts-56. After 13 passages the morphology of the infected cells was changed and later a cell line was established. Some features of this cell line are described and compared with a non-infected cell culture.

Evidence for peripheral mechanisms inducing tissue tolerance during ontogeny.

Tissue grafts from a histoincompatible donor of the same developmental stage were introduced into an early chick embryo host in order to probe the immune response to the graft after birth, when the host has reached immune maturity. Limb buds from B4 or B12 chicken strains were grafted in situ on (B15 x B21)F1 recipients that were allowed to hatch. The grafted wing grew normally and was tolerated in a nearly perfect way during the host's lifetime, although reversible rejection crises severely affected the fundamentally healthy state of the grafted tissues. Skin grafts of the same major histocompatibility complex haplotype as the wing were performed on the adult wing-chimera and were permanently tolerated. In contrast, host peripheral blood lymphocytes maintained their capacity to proliferate against donor cells in the mixed lymphocyte reaction. These results, while showing that in vitro and in vivo tolerance are separable phenomena, suggest the existence of a peripheral mechanism inducing tolerance to self that complements the elimination of self-reactive clones by the thymus.

[The family of env genes of avian retroviruses: molecular analysis of Rous sarcoma virus adapted to duck cells]. / Semeistvo genov env ptich'ikh retrovirusov: molekuliarnyi analiz virusa sarkomy Rausa, adaptirovannogo k utinym kletkam.

For the elucidation of the molecular basis of RSV adaptation to conditionally permissive host from the genome library of duck embryo fibroblasts, transformed by Rous sarcoma virus in 30 passages on these cells, recombinant bacteriophages that include provirus sequences, were obtained. Complete and transformation-defective proviruses were characterized, nucleotide sequences of their env-genes were compared with their counterparts the original RSV (Pr-RSV-C) and with viruses of other subgroups (A, B, D and E). The possible relation of the revealed changes in domains coding gp85 and gp37, with the changes of chicken RSV characteristics during adaptation to duck cells is discussed.

Detection of antibodies against hepatitis B core antigen using the avidin-biotin system.

An enzyme avidin-biotin assay for the detection of anti-HBcAg antibody in human sera was developed. The assay uses genetically engineered HBcAg. HBcAg is immobilized on the surface of the wells of microtitre plates and the test serum sample, biotin-labelled HBcAg and streptavidin-labelled horseradish peroxidase are added. The assay was found to be specific and was compared with a commercial radioimmunoassay kit for sensitivity by testing 96 human clinical sera for anti-HBcAg antibody. Both assays gave identical results.

Major histocompatibility complex class II (Ia-like) antigens on chicken embryo fibroblasts: effect of transformation by Rous sarcoma virus.

In this study, three monomorphic monoclonal antibodies to chicken MHC class II molecules (B-L) were tested for reactivity in normal and RSV-transformed embryo fibroblasts. The immunocytochemical staining, the cell-bound ELISA assay, and the immunoprecipitation analysis showed that all three antibodies reacted with the B-L (Ia-like) molecules on normal cells of different genotypes. Conversely, the expression of these antigens was not detected in fibroblasts cultured from feather follicles of adult birds. The level of expression of B-L molecules as well as the class II specific RNA increased consistently after transformation of the cells by the SR-RSV and infection with the avian leukosis virus RAV-1. Analysis of genomic DNA by the Southern blot technique, performed after digestion with several restriction endonucleases, showed that the restriction pattern of B-L genes was not altered in cells transformed by Rous sarcoma virus.

Expression of hepatitis B virus large envelope protein in Escherichia coli and Saccharomyces cerevisiae.

The gene coding for hepatitis B large envelope protein was cloned under the lac promoter in bacterial vector pUC-8 and under the ADH1 promoter in yeast expression shuttle vector pVT103-U, and expression of HBsAg in bacteria and yeast was determined. The strongest expression of large envelope protein was obtained after transformation of the protease-deficient yeast strain BJ1991. The recombinant large envelope protein did not form complex 22-nm particles and was not secreted into medium.