Michael Torten

Development of IL-2-independent feline lymphoid cell lines chronically infected with feline immunodeficiency virus : importance for diagnostic reagents and vaccines

Two interleukin 2 (IL-2)-independent feline immunodeficiency virus (FIV) producer cell lines (FL-4 and FL-6) were produced by selecting cells from an IL-2-dependent culture of mixed peripheral blood lymphocytes infected with FIV. The new cell lines have been stable for over 1 year and spontaneously produce FIV with an average reverse transcriptase titer of 300,000 cpm/ml and an average sucrose gradient purified viral protein concentration of 1 mg/l. FIV produced from these cultures is highly infectious in vitro and in vivo. The FL-6 cell line was phenotyped as expressing the feline CD8 and Pan-T antigens, while the FL-4 cell line expressed the CD4, CD8, and Pan-T antigens. Both cell lines, however, express high levels of viral core and envelope proteins. Paraformaldehyde-inactivated whole virus and similarly inactivated whole-cell virus preparations induced a strong antibody response to core and envelope antigens in immunized cats. The establishment of FIV-producing feline IL-2-independent peripheral blood lymphocyte lines should be valuable for the development of FIV-diagnostic reagents and vaccines and also as a model for human acquired immunodeficiency syndrome vaccine development.

Chemoimmunotherapy for canine lymphosarcoma

Thirty‐two dogs with naturally occurring multicentric lymphosarcoma were randomly assigned to one of two treatment groups. One half of the animals received combination chemotherapy plus vitamin injections (controls) while the other half received identical chemotherapy plus injections of chemically‐modified tumor cell extract in Freunds complete adjuvant (vaccinates). Clinical staging revealed no bias between groups but showed that prognosis could be closely correlated with the severity of disease at initial presentation. Twenty dogs (62%), including 11 vaccinates and 9 controls, responded favorably to chemotherapy and were evaluated for length of first remission and total survival time. Both parameters were significantly longer in vaccinated dogs than in controls. These data suggest that immunological stimulation may be a helpful adjunct to conventional therapy in selected types of cancer when immunological principles are observed.

ras oncogene mutations in diethylnitrosamine-induced hepatic tumors in medaka (Oryzias latipes), a teleost fish ☆

Medaka fish are an established non-mammalian research model for the study of liver carcinogenesis and exposure to environmental pollutants. Studies have emphasized the development of hepatic neoplasms in medaka following exposure to model carcinogens. To date however, little information is known regarding the mechanisms underlying initiation of hepatic tumors in this species. The aim of this study was to relate our understanding of diethylnitrosamine (DEN)-induced tumor formation to ras gene activation in hepatic neoplasms of exposed medaka. Initial studies were conducted to identify medaka ras exons 1 and 2 by reverse transcriptase polymerase chain reaction (RT-PCR). Amplification of ras exons 1 and 2 from untreated medaka liver resulted in the identification of three polymorphic ras sequence variants exhibiting a high degree of homology to other teleost and mammalian ras genes. Exposure of medaka to 159 ppm of DEN resulted in a wide range of hepatic neoplasms including: hepatocellular adenomas, hepatocellular carcinomas, cholangiomas, and mixed hepatocholangiocellular carcinomas. Individual liver tumors were examined for oncogenically activating ras mutations by probing genomic DNA with probes specific for activating point mutations or by direct cloning and sequencing of ras transcripts using RT-PCR. Using allele-specific oligonucleotide (ASO) analysis, a single point mutation was detected in codon 12 position two in 8/25 (32%) tumors examined. Mutated ras alleles were additionally detected in 12 of 39 (30%) medaka liver tumors by sequence analysis. Ten of the 12 mutations identified contained a single point mutation at codon 12 resulting in a Gly to Asp amino acid substitution. Two unique mutations were identified at codon 16 resulting in either Lys to Asn or Lys to Thr amino acid substitutions. Our results show that ras mutations are induced by DEN and are present in over 30% of the fish that developed tumors. A ras mutation incidence of 30% is similar to that reported in mammalian species exposed to DEN. While mutations at codon 12 have previously been reported, the present study is the first in vivo report of ras point mutations at codon 16.

Evaluation of in vivo and in vitro interactions of feline immunodeficiency virus and feline leukemia virus

Objective:To determine the potential mechanisms for disease potentiation where feline immunodeficiency virus (FIV) infection of persistently feline leukemia virus (FeLV)-infected cats results in more severe FIV disease and increased mortality than FIV infection of specific pathogen-free cats. Design and methods:To determine whether pseudotype formation resulting in expanded cell tropism may be an important mechanism, cellular targets and tissue distribution of FIV and FeLV were determined by in situ hybridization and/or immunohistochemistry. To determine whether FeLV can transactivate the FIV long terminal repeat (LTR) resulting in increased FIV expression, in vitro transient expression assays were performed. To examine whether persistent FeLV infection can cause the deletion of a suppressive T-lymphocyte population, peripheral blood mononuclear cell (PBMC) cultures from persistently FeLV-infected cats were infected with FIV and monitored for FIV antigen levels.Results:Macrophages were the predominant target of FIV infection and were disseminated in a similar pattern in lymphoid and nonlymphoid tissues of both FIV-infected and FeLV/FIV-coinfected cats. FeLV-infected cells expressing FIV RNA were not present. Significant transactivation of the FIV LTR in FeLV-infected cells was not demonstrated. FIV antigen production was similar upon in vitro infection of PBMC from FeLV-infected and uninfected cats.Conclusions:Neither direct virus/virus interactions, such as FeLV/FIV pseudotype formation or transactivation of the FIV LTR in FeLV-infected cells, nor deletion of a regulatory cell subset from the blood of FeLV-infected cats, was found to be the mechanism of disease potentiation.

TUMOR VACCINES FOR IMMUNOTHERAPY OF CANINE LYMPHOSARCOMA

Canine hemolymphatic disease is a group of neoplasms that are frequently encountered in the veterinary practice. Priester and Mantel l reported that it was the fourth most common neoplasm in the dog. Lymphosarcomas (malignant lymphoma) comprise the majority of hemolymphatic tumors, whereas granulocytic leukemia is relatively rare in the dog. Dorn et aL8 reported an incidence of lymphosarcoma of 241 100,000 dogs in a defined population in two California counties. There is increased frequency of the disease with age. Canine malignant lymphoma is usually seen as a generalized lymphatic disease. In a series of 146 dogs with lymphosarcoma from our clinic, 84% had a generalized lymph node involvement. The liver and spleen were involved, respectively, in 54 and 62% of all the animals. Involvement of just the organs of the abdomen or of the thorax or the skin accounts for 14% of our 146 cases. Most of the lymphosarcoma animals presented to our clinic are in advanced stages of disease, usually in stages I11 and IV according to the criteria of Squire et aL3 Until recently, lymphosarcoma was classified as an untreatable fatal disease of dogs. There are little data in the literature that provide information that pertains to spontaneous remissions and to survival after diagnosis in untreated dogs. Unfortunately, most dogs with lymphosarcoma are euthanized, so data from the literature and from our own experience with untreated animals are scarce. Nevertheless, it is recognized that without treatment, dogs rarely survive beyond 30 days after clinical diagnosis (TABLE 1). A report from Scotland indicated a longer duration of life, but the survival time was calculated from the time the owners first noted signs of illness rather than from time of established diagnosis by a veterinarian. However, even there, the mean survival time was less than 2 months. Of 91 dogs presented to our clinic with lymphosarcoma and not treated, owners requested that 5 1 be immediately euthanized. Seventeen were hospitalized for clinical work-up. These dogs died with a mean time of 2.5 days. Seventeen other dogs in less advanced disease were sent home without treatment. The latter group survived from 5 to 133 days, with an approximate mean of 30 days (TABLE 1 ) .

Induction of ras oncogene mutations and hepatocarcinogenesis in medaka (Oryzias latipes) exposed to diethylnitrosamine

Abstract Medaka ( Oryzias latipes ), a fish model for assessing the carcinogenic impact of environmental exposure to genotoxic chemicals, were subjected to 390 ppm of diethylnitrosamine (DEN) in an aqueous bath for 48 h. Histological examinations, at 5 and 7 months post-exposure, revealed that 11 of 17 fish developed hepatic neoplasms (hepatocellular carcinomas, cholangiomas and mixed hepato-cholangiocellular carcinomas). Genomic DNA extracted from the livers of normal medaka was used to obtain and sequence the structure of ras gene exon 1 and exon 2. Comparing PCR-amplified products from exon 1 and 2 by hybridization with specific mutation containing probes and/or by sequencing of liver DNA from control and exposed fish revealed an association of ras oncogene mutations and DEN-induced tumor formation. While none of the control fish showed any mutations, three of 11 fish with tumors had ras gene mutations. All three fish had mutations at codon 12 replacing glycine with aspartic acid, while one fish had an additional mutation at codon 16 replacing lysine with threonine. In addition to identifying DEN-induced mutations at codon 12 of the medaka ras oncogene, the present study represents a first report of an in vivo association of hepatocarcinogenesis, in any species, with a mutation at codon 16. Mutation at codon 16 and tumorigenesis has been previously predicted and artificially produced. Our study also supports the relevance of the medaka fish model for in vivo detection of the carcinogenic potential of environmental pollutants in sediments and biota of ambient water systems.