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Biomedical subjects

C Altaner

Publications and source records attributed to C Altaner.

At least 37 records · Page 2Linked to original sources
PubMed1997

Differential display of RNA from tumorigenic and nontumorigenic variants of hamster cells transformed with avian sarcoma virus.

Differential display technique was applied to study expression of RNA in tumorigenic and nontumorigenic cell variants of avian sarcoma virus transformed hamster cells. Methodical conditions were worked out, which allowed identifying a cDNA fragment of an unknown gene expressed in nontumorigenic cell variant only. Its role in tumor suppression remains to be determined.

Animals↗
PubMed1997

Treatment of rat gliomas with recombinant retrovirus harboring Herpes simplex virus thymidine kinase suicide gene.

The retrovirus vector containing Herpes simplex virus type 1 thymidine kinase (HSVtk) gene was constructed. The vector was transfected into the packaging cell line PG13. It was shown that individual transfected cells differ in the production of recombinant retrovirus and in their susceptibility to be killed by ganciclovir. Recombinant retrovirus with a gibbon envelope was able to transduce the HSVtk gene into rat glioma cells. In vivo studies confirmed the ability of intraperitoneal ganciclovir administration to influence subcutaneous and intracerebral tumors developed after injection of C6 rat glioma cells with subsequent injection of HSVtk retrovirus producing cells.

Animals↗
PubMed1995

Gene therapy for cancer (present status).

The present status of cancer gene therapy is reviewed here in short. Two of the main gene therapy strategies for the treatment of cancer are discussed. The first main strategy is direct gene therapy which involves insertion of a functioning tumor suppressor gene or suppression of expression of a known oncogene. The second main strategy is indirect gene therapy which involves the insertion of a gene that modifies the cell to be more immunogenic for the host. The main clinical gene therapy trials are reviewed in their present state, including the replacement of defective tumor suppressor genes, the insertion of suicide or sensitivity genes, the insertion of prodrug-activating genes, and the use of virally directed enzyme prodrug therapies. Other topics discussed are the protection of stem cells from toxic effects of chemotherapy and new directions for gene therapy of neoplastic disease.

Clinical Trials as Topic↗
PubMed1994

Isolation of the missing 5'-end of the encoding region of the bovine leukemia virus cell receptor gene.

The missing 5'-end of the encoding region of the bovine leukemia virus (BLV) cell receptor gene (BLVRcp1/5') was isolated from a lambda gt11 cDNA library using the 32P-labeled EcoRI-SamI fragment corresponding to the 5'-end of a 2.3 kbp cDNA fragment encoding the binding domain of the bovine leukemia virus cell receptor gene (BLVRcp1). The nucleotide and amino acid sequence analysis of the BLVRcp1/5' cDNA revealed that the 1058 bp EcoRI fragment at its 5'-end contained a new 114 amino acid long sequence, and at its 3'-end contained a completely identical 88 amino acid overlapping region with the 5'-end of the BLVRcp1 cDNA. The combined sequences of both cDNAs represent the whole encoding region of the BLV cell receptor gene. The longest open reading frame of the BLV cell receptor gene encodes a protein containing 843 amino acids with a calculated molecular mass of 94.2 kDa which concurs with experimentally detected native BLV receptor protein. Search for homology has shown that about 250 bp of the BLV cell receptor gene is highly homologous to Venter's tag sequences of an unidentified gene from the human brain library.

Amino Acid Sequence↗
PubMed1993

Envelope glycoprotein gp51 of bovine leukemia virus is differently glycosylated in cells of various species and organ origin.

The carbohydrate moiety of the envelope glycoprotein gp51 of bovine leukemia virus, American strain, was studied. The virus was grown in ovine, bovine, porcine, bat and rat cells of various organ specificities. The gp51 was purified by immunoaffinity chromatography from virions of ten different virus-producing cells derived from various body organs of different species. Highly purified glycoproteins (single band in PAGE) were compared for their electrophoretic mobility, for the presence of epitopes by a battery of monoclonal antibodies, and for the glycosylation pattern by lectin blot analysis. Electrophoretic analysis of all tested glycoproteins deglycosylated by glycopeptidase F detected the same polypeptide backbone according to PAGE. The glycoproteins produced in rat cells migrated faster in PAGE, as detected in cells or in virions, than those produced in ovine cells. The pattern of their glycosylation was found to be dependent on the type of cells used for virus production. The differences in glycosylation were most pronounced when comparing the glycoprotein produced in ovine cells versus bat or rat cells. Changes in epitope expression were also detected. The differences in the patterns of glycosylation and in the accessibility of epitopes owing to the virus production in various kind of cells are discussed from virus infectivity and vaccine points of view.

Animals↗
PubMed1993

Two immunodominant regions revealed by monoclonal antibodies on the main structural protein p24 of bovine leukemia virus.

Eleven different monoclonal antibodies (Mabs) directed against the main structural protein p24 of bovine leukemia virus (BLV) were prepared. All Mabs reacted with p24 in Western blot and in radioimmunoprecipitation. Competition antibody binding assays with the prepared Mabs distinguished three independent groups of Mabs. Two immunodominant regions (IDRs) of p24 BLV were defined by these Mabs. The Mabs were induced preferentially against two immunodominant regions on the native form of p24 BLV (BLVp24 IDR-1 and BLVp24 IDR-2). Mab of the third group was directed against a different immunogenic epitope of p24 BLV. A model of the IDRs based on the differences in the fine epitope specificity of Mabs defining these immunodominant regions is proposed.

Animals↗
PubMed1993

Isolation and characterization of a 2.3-kilobase-pair cDNA fragment encoding the binding domain of the bovine leukemia virus cell receptor.

An immunoscreening strategy was used to isolate a cDNA clone encoding the binding domain for the external glycoprotein gp51 of the bovine leukemia virus (BLV). Three recombinant phages demonstrating BLV binding activity and containing 2.3-kbp cDNA inserts with identical nucleotide sequences were isolated from a lambda gt11 cDNA library of bovine kidney cells (MDBK). One clone, BLVRcp1, hybridized with a 4.8-kb mRNA from cells of bovine origin and was also found to be conserved as a single-copy gene in murine, bovine, ovine, primate, canine, feline, and porcine DNAs. The same gene is amplified in caprine DNA isolated from a BLV-induced tumor. The longest open reading frame of BLVRcp1 encodes a protein fragment of 729 amino acids with a putative receptor structure. BLVRcp1 cDNA was cloned in the eucaryotic expression vector pXT-1 and transfected into murine NIH 3T3 and human HEp-2 cells. Cells expressing BLVRcp1 mRNA became susceptible to BLV infection. BLVRcp1 has no known physiological function and has no significant homology with sequences registered in the GenBank and EMBL data libraries (31 July 1992). Expression of deleted constructs of BLVRcp1 indicates that the BLV binding region is encoded at the 5' side of the receptor clone.

Amino Acid Sequence↗
PubMed1993

Preparation and characterization of monoclonal antibodies directed against glycoproteins of bovine leukaemia virus.

Six monoclonal antibodies (MoAbs) directed against glycoproteins of bovine leukaemia virus (BLV) were prepared and characterized. Comparison of these MoAbs with anti-gp51 MoAbs of known epitope specificity by competition antibody binding assay allowed to distinguish two new conformational epitopes C1 and C2 on the molecule of gp51. The epitope C1 is involved in the process of inhibition of formation of syncytia but not in neutralization of VSV/BLV pseudotypes. Three newly prepared MoAbs were directed against known epitopes F, G and H, and their neutralizing activities of biological functions of gp51 were determined. MoAbs BLVgp30-94C11 which was directed against transmembrane glycoprotein gp30 was found not to be involved in neutralization of VSV/BLV pseudotypes and did not inhibit formation of syncytial cells as well.

Animals↗
PubMed1992

Mapping of sequential epitopes recognized by monoclonal antibodies on the bovine leukaemia virus external glycoproteins expressed in Escherichia coli by means of antipeptide antibodies.

A lambda gt11 cDNA library prepared from bovine leukaemia virus (BLV)-producing ovine cells was screened with a cocktail of anti-BLV gp51 monoclonal antibodies (MAbs). Four recombinant phages with inserts of about 2-5 kbp were isolated. One, lambda BLV-gp51-1, was sequenced and shown to encode the C-terminal part of gp51 and all of gp30. This insert was subcloned into pEV-vrf1 and expressed in Escherichia coli N-4830-1 cells. The BLV product and a series of antipeptide antibodies were used to localize the sequential epitopes defined on BLV envelope glycoprotein gp51 by their reactivity with MAbs. Epitope B was localized to amino acids 180 to 205, B' to residues 195 to 205, D and D' to residues 218 to 237, and A to amino acids 249 to 260. All the mapped sequential epitopes were localized in the C-terminal half of BLV gp51. The results of epitope mapping with bacterially produced gp51 confirm the map obtained using native viral glycoprotein.

Amino Acid Sequence↗
PubMed1991

Protective vaccination against bovine leukaemia virus infection by means of cell-derived vaccine.

Tests were performed to determine whether live mammalian cells producing env gene glycoproteins and main structural protein p24 of bovine leukaemia virus (BLV), heterologous to bovine species, could serve as an immunogen in cattle to prevent induction of bovine leukaemia. Ovine virus-non-producing clonal cells NP-2 were used as the immunogen. The NP-2 cells synthesized only the env gene products--glycoprotein gp51 and gp30 and main structural protein p24 of BLV. The NP-2 cells, inoculated into rats, induced an antibody response directed against envelope glycoproteins of BLV. The antibodies neutralized the infectivity of BLV as determined by the VSV/BLV pseudotype neutralization test. Similar results were obtained by vaccination of cattle with these cells. A dose of less than or equal to 2 x 10(6) live cells inoculated subcutaneously induced an antibody response in cattle, while a high dose of killed cells was ineffective. The antibodies in cattle were directed against env products of BLV. A group of 92 cows was vaccinated and followed up for 4 years. The antibody levels fluctuated slightly during the 4-year observation period, generally decreasing with time, but revaccination always increased the antibody titre. No transfer of seropositivity was observed to seronegative animals which were kept in contact with vaccinated ones. In a separate experiment a group of young heifers, after repeated vaccination, were challenged with a high dose of infectious virus and/or virus-producing cells. The response to BLV infection was followed by syncytial induction assay after co-cultivation of white blood cells with indicator cells CC81.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Antigen capture assay for detection of bovine leukemia virus proteins by monoclonal antibodies.

A capture monoclonal antibody-based assay has been established for detecting the p24 core protein and the gp51 envelope glycoprotein of bovine leukemia virus (BLV). This assay is rapid, highly sensitive and specific. Viral antigens in test samples were identified using mouse monoclonal antibody-coated or microtiter plates by adding labeled monoclonal antibodies with different epitope specificities. The choice of an appropriate epitope specificity for the specificity of monoclonal antibodies was important for optimal performance of the assay. Results of this assay were in agreement with the syncytia induction assay routinely used for detecting BLV production by cells in vitro. The sensitivity of monoclonal antibody assay was 0.5 ng/ml for p24 and 1.25 ng/ml for gp51, respectively. The specificity was demonstrated by immunoblotting. The assay can be performed in a few hours, is simple, and is comparable with more time-consuming assays with regard to sensitivity and specificity.

Animals↗
PubMed1990

Use of monoclonal antibodies in an ELISA for the diagnosis of bovine leukaemia virus infection.

An ELISA diagnostic test for detection of bovine leukaemia virus (BLV) infected animals was developed. The test is based on the use of a mixture of monoclonal antibodies (MAbs) against envelope glycoprotein and against viral structural protein p24. The sensitivity and specificity of the test were found to be dependent on the relative proportions of MAbs of the appropriate epitope specificity. Polystyrene microtitre plates, wells or sticks were firstly coated with a mixture of purified MAbs and then non-purified viral antigens were adsorbed from tissue culture fluid obtained from BLV-producing cells. The optimal conditions for adsorption of MAbs and viral antigens as well as for the ELISA procedure were established. The test is more sensitive and cheaper (no need for virus antigen purification) than the routinely used ELISA using purified virus antigens. The assay is highly specific, rapid, practical and could be easily automated. It is suitable for the detection of BLV-antibodies in blood serum or milk in the large-scale screening programs for BLV-infected animals.

Animals↗
PubMed1990

Induction of leukemia in chicken by bovine leukemia virus due to insertional mutagenesis.

Bovine leukemia virus (BLV) was inoculated into one-day-old chickens. In a small part of inoculated chickens leukemia developed during observation period of one year. Out of 88 birds inoculated, only 4 developed histopathologically verified leukemia. The induced leukemia was characterized by enlarged liver and spleen. The organs were infiltrated with leukemic cells. The DNAs of body organs of inoculated chickens were analysed by Southern blot hybridization for the presence of BLV specific sequences. Out of 9 suspicious chickens tested in 6 birds the BLV was found to be integrated into host DNA either as a complete viral genome or as a part corresponding to its 3'-end. The leukemic cells were monoclonal as regard to the integration site of the BLV provirus. Neither the expression of BLV provirus in chicken leukemic cells nor the antibody response to BLV antigens in inoculated birds was detected. The rearrangements and amplification of erb-B and myb loci of protooncogenes in leukemic cells was detected. There were no changes in loci of following protooncogenes: myc, sis, fes, fps, erb A, src and yes. All obtained data taken together suggest that the BLV induced leukemia in chickens is caused by insertional mutagenesis.

Animals↗
PubMed1989

Induction of immune deficiency syndrome in rabbits by bovine leukaemia virus.

Newborn rabbits were inoculated with bovine leukaemia virus (BLV). The majority of infected rabbits produced antiviral antibodies. All the seroconverted animals developed symptoms resembling AIDS and died several months after inoculation. The course of experimental infection of rabbits with BLV resembled in many respects the broad spectrum of clinical disorders associated with AIDS induced by HIV. Antibody response to virus proteins was followed by immune deficiency and signs of neuropathy, and the animals subsequently died of opportunistic infections. Virus transmission from infected babies to the mothers by contact was also observed. In some cases the virus was salvaged from lymphocytes of rabbits with the immune deficiency syndrome. The virus-specific sequences were found to be integrated at random in the DNA of haematopoietic cells and of some organs. Slight expression of viral RNAs in lymphocytes was found. Experimental infection of rabbits with BLV can be used in experiments to understand AIDS induction.

Acquired Immunodeficiency Syndrome↗
PubMed1989

Infection of rats with bovine leukaemia virus: establishment of a virus-producing rat cell line.

Adult rats were infected with bovine leukaemia virus (BLV). Inoculated rats persistently produced antibodies directed against viral structural proteins. No major pathogenesis in infected rats was found during 2 years of observation. It was possible to recover the virus from rat spleen several months after infection. A cell line, R(BLV), was established from rat spleen; this contained integrated BLV provirus. R(BLV) cells kept for over 80 passages in vitro produced viral particles with the properties of BLV. Provirus reintegration and/or amplification occurred in R(BLV) cells. The cell line was found to be tumorigenic in rats, and the virus produced was immunogenic. R(BLV) cells represent the first described BLV-producing rat cell line. Proven persistent infection with BLV indirectly suggests that rats can serve as a reservoir of BLV in nature.

Animals↗
PubMed1989

Human cells of neural origin are permissive for bovine leukemia virus.

Bovine leukemia virus (BLV) propagated in a cell clone of fetal lamb kidney origin was transmitted by cell contact to different mammalian cells including human cells. The transmission of the BLV genome was effectively achieved by cocultivation of mitomycin-C-killed, virus-producing cells of the cell clone with recipient cells. In particular, human cells of neural origin were highly susceptible to BLV infection, while some other cells were resistant. The transmission of the BVL genome from virus-nonproducing cells failed which suggests the existence of virus specific receptors on the cells. The donor cells contained three integrated BLV proviruses. In recipient cells only one provirus was found. The majority of cells contained both unintegrated and integrated BLV provirus. In the cells containing the transmitted BLV, the viral genome was expressed to its protein products. The results indirectly suggest that retroviruses with similar properties could cause various neural diseases in man.

Animals↗
PubMed1988

Modifications of hybridoma technology which improve the yield of monoclonal antibody producing cells.

Several modifications at various stages of the standard hybridoma technique were found to increase the yield of monoclonal antibody-producing cells. Lymphocytes obtained from draining lymph nodes of mice immunized over a 10 day period with antigen injected into the foot pads were used for cell fusion. Preincubation of myeloma cells with lymphocytes in the presence of 0.25% polyethylene glycol at 37 degrees C for 90 min increased the yield of antibody-secreting hybrid colonies ten times. The use of conditioned medium from cultivated rat thymocytes ('lymphokines') as a supplement to cultivation medium made it unnecessary to use feeder cells, and increased the growth rate of the hybridomas. No change of the culture fluid was needed during the time which was necessary to grow up the cells to be tested for monoclonal antibody production. By a combination of the described procedures, the time required from the start of immunization to the screening for positive hybridomas was shortened to 23 days.

Animals↗