Cellular, serological, and molecular polymorphism of the class I and class II loci of the canine Major Histocompatibility Complex.

This study was undertaken to determine the relationships between canine cellular and serological determinants and more recently described genes. Such relationships might reveal information about immunological reactivity or function of various proteins. To do this we studied the haplotypic associations of dog leukocyte antigen (DLA) class I and class II alleles determined from a panel of 14 DLA-D homozygous dogs. This panel of dogs was typed for the serological determinants DLA-A, DLA-B and DLA-C. Polymorphisms for DLA-DQA1, DLA-DQB1, DLA-DRB1 and DLA-88 were also determined. The number of alleles (one or two) for two microsatellite markers in the DLA region were also determined. Analyses of the nucleotide sequences and of the serological and cellular typing data revealed that phenotypic homozygosity, as defined by the DLA-D type in mixed leukocyte culture (MLC), tended to correlate with homozygosity at the DLA-DRB1 locus but not necessarily at the DLA-DQB1 locus. Furthermore, MLC specificity was determined by other loci besides DLA-DRB1 and DLA-DQB1. The amino acid at position 63 of the DR beta chain could contribute to the DLA-B serological specificity. DLA-88, the most polymorphic class I gene characterized to date, did not have an easily identifiable association with either the DLA-A or DLA-C class I serological specificities. Homozygosity or heterozygosity of each of two microsatellite markers, FH 2200 and FH 2202, located in the class I or class II region, respectively, did not correlate with homozygosity or heterozygosity of the most polymorphic known class I (DLA-88) or class II (DLA-DRB1) genes.

Biologic effects of recombinant human interleukin-12 in squirrel monkeys (Sciureus saimiri).

BACKGROUND: Interleukin-12 is a novel heterodimeric cytokine that stimulates the proliferation of activated T and NK cells and induces lymphokine-activated killer cell activity in vitro. To investigate the biological effects of recombinant human IL-12 (rHuIL-12) in vivo, two exploratory studies were conducted in squirrel monkeys (Sciureus saimiri), which have been shown to be pharmacologically responsive to rHuIL-12 in vitro. EXPERIMENTAL DESIGN: In the first study, 18 monkeys (3/sex/group) were given daily subcutaneous injections of 0 (vehicle control), 10, or 50 micrograms/kg/day rHuIL-12 for 14 days. In the second study, 18 monkeys were given 0, 0.1, or 1 micrograms/kg/day rHuIL-12 for 14 days The animals were monitored for clinical signs, hematology and clinical chemistry changes, and sacrificed on day 15 to evaluate gross and histopathologic changes. One monkey in the high dose group was sacrificed moribund on day 14. RESULTS: Monkeys given rHuIL-12 had dose-related hematologic changes characterized by mild to moderate anemia and leukocytosis. Serum chemistry changes included hypoproteinemia, hypoalbuminemia, hypophosphatemia, and hypocalcemia. Gross pathologic findings included generalized lymph node enlargement and splenomegaly with pulmonary edema and peritoneal effusions in two high dose monkeys. Dose-related histopathologic findings included thymic cortical atrophy, splenic lymphoid hyperplasia with histiocytic hyperplasia and extramedullary hematopoiesis of red pulp, Kupffer cell hypertrophy and hyperplasia, trilineage bone marrow hyperplasia, and reactive hyperplasia of lymph nodes. Animals in the 10 and 50 micrograms/kg/day dose groups developed high titers of anti-rHuIL-12 antibodies by day 15. CONCLUSIONS: These studies indicate that rHuIL-12 is bioactive over a wide dose range and induces prominent hyperplasia of hematopoietic and lymphohistiocytic tissues in squirrel monkeys. Moreover, positive immunomodulatory activity (enhanced lymphocyte lytic activity) was detected at a dose of rHuIL-12 that is 500-fold less than the dose causing severe toxicity.

In vivo toxicological effects of rel A antisense phosphorothioates in CD-1 mice.

To characterize the in vivo toxicity of phosphorothioate antisense oligonucleotides against rel A (p65 subunit of NF-kappa B transcription factor), forty-eight 6-week-old CD-1 mice were split into 4 groups (6/sex/group) receiving vehicle (phosphate-buffered saline) or doses of 50, 100, and 150 mg/kg of rel A antisense oligonucleotides intraperitoneally 3 times weekly for 2 weeks. Clinical signs of toxicity included weakness, and decreased motor activity and food consumption with body weight loss. Mortality occurred in 7 of 12 mice in the 150-mg/kg group and in 2 of 12 mice in the 100-mg/kg group, most of which died within the first 2 to 4 days of treatment. The remaining mice were necropsied on day 15. The major hematological finding was severe dose-dependent thrombocytopenia. The liver enzyme levels were mildly elevated in the serum of mid- and high-dose animals. At necropsy, increased spleen and liver weights were observed in treated mice, some of which also had mild pleural and/or peritoneal effusions. Histopathological examination revealed the likely cause of death to be acute renal failure due to renal cortical or tubular necrosis. Treatment-related changes were also found in the liver, spleen, bone marrow, and several other organs. In summary, the kidney, liver, and bone marrow (megakaryocytic lineage) were identified as the major target organs for toxicity with rel A antisense therapy.

Antisense inhibition of the p65 subunit of NF-kappa B blocks tumorigenicity and causes tumor regression.

The NF-kappa B transcription factor, composed of two proteins, p50 and p65, is a pleiotropic activator that participates in the induction of a wide variety of cellular genes. Various cell adhesion molecules have NF-kappa B binding sites and may play an important role in inflammatory response, tumorigenicity, and metastasis. In an earlier study, we demonstrated that adhesion of diverse transformed cells was blocked by antisense inhibition of the p65 subunit of NF-kappa B. Since cell-substratum interactions play an important role in tumorigenicity, we reasoned that antisense p65 could inhibit tumorigenicity. In diverse transformed cell lines, phosphorothioate antisense oligonucleotides to p65 inhibited in vitro growth, reduced soft-agar colony formation, and eliminated the ability of cells to adhere to an extracellular matrix. Stable transfectants of a fibrosarcoma cell line expressing dexamethasone-inducible antisense RNA to p65 showed inhibition of in vitro growth and in vivo tumor development. In response to inducible expression of antisense RNA, a pronounced tumor regression was seen in nude mice. The administration of antisense but not sense p65 oligonucleotides caused a pronounced inhibition of tumorigenicity in nude mice injected with diverse tumor-derived cell lines. Inhibitors of NF-kappa B function may thus be useful in the treatment of cancer.

A sense phosphorothioate oligonucleotide directed to the initiation codon of transcription factor NF-kappa B p65 causes sequence-specific immune stimulation.

Antisense oligonucleotides have proved effective in achieving targeted inhibition of gene expression. In such experiments, sense oligonucleotides have frequently been used as a control for nonspecific effects, but the results have been variable, raising questions about the reliability of sense oligomers as a control. It is possible that some of the effects of sense oligonucleotides may be specific. We have shown that phosphorothioate antisense oligonucleotides to the p65 subunit of NF-kappa B, a transcription factor, cause a block in cell adhesion. In our efforts to test the efficacy of NF-kappa B p65 oligonucleotides in vivo, we unexpectedly observed that the control p65-sense, but not the p65-antisense, oligonucleotides caused massive splenomegaly in mice. In the current study we demonstrate a sequence-specific stimulation of splenic cell proliferation, both in vivo and in vitro, by treatment with p65-sense oligonucleotides. Cells expanded by this treatment are primarily B-220+, sIg+ B cells. The secretion of immunoglobulins by the p65-sense oligonucleotide-treated splenocytes is also enhanced. In addition, the p65-sense-treated splenocytes, but not several other cell lines, showed an upregulation of NF-kappa B-like activity in the nuclear extracts, an effect not dependent on new protein or RNA synthesis. These results demonstrate that phosphorothioate oligonucleotides can exert sequence-specific effects in vivo, irrespective of sense or antisense orientation.

Allelic variation in the DR subregion of the canine major histocompatibility complex.

Allelic variation in the DR subregion of the canine major histocompatibility complex (DLA) has been analyzed by nucleic acid sequencing of cDNA clones of DRB genes amplified in vitro by the reverse transcriptase-polymerase chain reaction (RT-PCR). Sequence analysis of a panel of 19 homozygous typing cell dogs representing 12 different DLA-D types (defined by mixed leucocyte reaction) demonstrated the presence of one expressed DRB locus with at least nine distinct alleles in the dog. Unique DLA-DRB alleles were found in the DLA-D types Dw1, Dw3, Dw4, Dw8 (workshop assignments) and D4, D6, D7, D8, and D9 (Seattle assignments). In contrast, the DRB genes of the remaining three DLA-D types (D1, D10, and D16) were identical to those of Dw3/Dw4 (for D1), Dw8 (for D10), and D6 (for D16). The nucleotide sequences of all nine DLA-DRB alleles were typical of functional major histocompatibility complex (MHC) class II beta chains and contained three allelic hypervariable regions (HVRs) in the beta 1 domain at positions 8-16, 26-39, and 57-74. At each variable residue, two to five amino acid substitutions were found. The most polymorphic residues were located at positions 37 (with five amino acid substitutions), 11, 13, 28, and 71 (each with four substitutions). The DLA-DRB alleles had 96%-99% overall nucleotide sequence similarity and 93%-99% amino acid sequence similarity with each other. Cluster analysis of the nucleotide and predicted amino acid sequences subdivided the DLA-DRB alleles into three major allelic groups which may represent the canine counterparts of the supertypic groups described in man.

RFLP analysis of DLA class I genes in the dog.

Human major histocompatibility complex (HLA) cDNA probes for class I genes HLA-A,B and HLA-E were used to analyze the Restriction Fragment Length Polymorphism (RFLP) of the class I region of the canine major histocompatibility complex (DLA) in 40 dogs. The Southern blot analysis demonstrated that the dog genome contains at least eight class I genes, including the canine homologues of HLA-A,B and HLA-E genes. The DNA polymorphism detected by the HLA-B7 probe corresponded to the serologically defined DLA-A allelic series. Restriction fragments that correlated with the DLA-A2, -A7 and -A9 antigenic specificities were identified in PstI digests of genomic DNA. The RFLP analysis was particularly useful in genotyping dogs which were not clearly DLA-A typable by serology. This technique can be used as a supplement to serotyping and as a genotyping tool for DLA antigenic specificities for which specific antisera are not available.

Characterization of class II alpha genes and DLA-D region allelic associations in the dog.

Human major histocompatibility complex (HLA) cDNA probes were used to analyze the restriction fragment length polymorphism (RFLP) of the alpha genes of the DLA-D region in dogs. Genomic DNA from peripheral blood leucocytes of 23 unrelated DLA-D homozygous dogs representing nine DLA-D types (defined by mixed leucocyte reaction) was digested with restriction enzymes (BamHI, EcoRI, Hind III, Pvu II, Taq I, Rsa I, Msp I, Pst I and Bgl II), separated by agarose gel electrophoresis and transferred onto Biotrace membrane. The Southern blots were successively hybridized with radiolabelled HLA cDNA probes corresponding to DQ, DP, DZ and DR alpha genes. Clear evidence was obtained for the canine homologues of DQ and DR alpha genes with simple bi- or tri-allelic polymorphism respectively. Evidence for a single, nonpolymorphic DP alpha gene was also obtained. However, the presence of a DZ alpha gene could not be clearly demonstrated in canine genomic DNA. This report extends our previous RFLP analysis documenting polymorphism of DLA class II beta genes in the same panel of homozygous typing cell dogs, and provides the basis for DLA-D genotyping at a population level. This study also characterizes the RFLP-defined preferential allelic associations across the DLA-D region in nine different homozygous typing cell specificities.

Bovine progressive degenerative myeloencephalopathy (weaver syndrome) in brown swiss cattle in Canada: a literature review and case report.

A 15-month-old purebred Brown Swiss heifer was presented because of posterior paresis and ataxia. Histopathological examination of the brain and spinal cord showed evidence of a mild diffuse degenerative myeloencephalopathy. The most severe degenerative lesions were located in the white matter of the thoracic spinal cord. We believe this to be the first documented case of bovine progressive degenerative myeloencephalopathy ("weaver syndrome") in Canada.

Restriction fragment length polymorphism of the major histocompatibility complex of the dog.

Human major histocompatibility complex (HLA) cDNA probes were used to analyze the restriction fragment length polymorphism (RFLP) of the DLA-D region in dogs. Genomic DNA from peripheral blood leucocytes of 23 unrelated DLA-D-homozygous dogs representing nine DLA-D types (defined by mixed leucocyte reaction) was digested with restriction enzymes (Bam HI, Eco RI, Hind III, Pvu II, Taq I, Rsa I, Msp I, Pst I, and Bgl II), separated by agarose gel electrophoresis, and transferred onto Biotrace membrane. The Southern blots were successively hybridized with radiolabeled HLA cDNA probes corresponding to DR, DQ, DP, and DO beta genes. The autoradiograms for all nine enzyme digests displayed multiple bands with the DRb, DQb, and DPb probes while the DOb probe hybridized with one to two bands. The RFLP patterns were highly polymorphic but consistent within each DLA-D type. Standard RFLP patterns were established for nine DLA-D types which could be discriminated from each other by using two enzymes (Rsa I and Pst I) and the HLA-DPb probe. Cluster analysis of the polymorphic restriction fragments detected by the DRb probe revealed four closely related supertypic groups or DLA-DR families: Dw3 + Dw4 + D1, Dw8 + D10, D7 + D16 + D9, and Dw1. This study provides the basis for DLA-D genotyping at a population level by RFLP analysis. These results also suggest that the genetic organization of the DLA-D region may closely resemble that of the HLA complex.

A canine lymphocyte surface antigen detectable by a monoclonal antibody (DT200).

A murine monoclonal antibody (DT200) was raised against a 210,000-dalton (210 K) lymphocyte surface protein (a member of the lymphocyte antigen known as T200) which was purified from a canine lymphoid tumor by preparative slab gel electrophoresis. In immunoblotting studies of electrophoretically separated plasma membranes from five cases of canine lymphoma, the antibody detected two antigenically intact peptides at 95 and 110 K which, based on previous polyclonal anti-210 K antiserum immunoblotting and peptide mapping studies, may represent the protease-resistant fragment of the canine T200 molecule. Since DT200 retains its reactivity in formalin-fixed, paraffin-embedded tissue sections, 13 dogs with malignant lymphoma and a panel of normal lymphoid and nonlymphoid tissues were studied using an indirect immunoperoxidase technique. The antigen was localized predominantly to the surface membrane of lymphoid cells. DT200 reacted strongly with all five histological subtypes of lymphoma tested while moderate reactivity was detected in normal B and T cell areas of lymph node, spleen and tonsil. Thymocytes and selected hemopoietic precursors were weakly reactive with DT200 while plasma cells, mature granulocytes, red cells and megakaryocytes were unstained. It was concluded that DT200 is a useful reagent for the diagnosis of malignant lymphoma particularly in extranodal sites and may prove valuable in the investigation of the structure and function of T200 in the dog.

Expression of the T200 family of glycoproteins in canine malignant lymphoma.

High molecular weight surface proteins were examined in lymph node lymphocytes from five control dogs and 27 dogs with malignant lymphoma. Polyclonal rabbit antiserum was raised against a 210,000-dalton (210 K) membrane protein which was purified from a canine lymphoid tumor by preparative slab gel electrophoresis. Three high MW proteins at 210, 195 and 170 K and a common proteolytic fragment at 95 K were detected in the electrophoretically separated plasma membrane preparations by immunoblotting with polyclonal anti-210 K antiserum. Two antigenically distinct patterns were evident: 1) Type 1 lymphomas expressed a major 210 K peptide (with or without a minor 195 K component), and 2) Type 2 lymphomas lacked the 210 K form but had a 170 K or 195 K peptide singly or in combination. Immunoperoxidase staining of formalin-fixed, paraffin-embedded tissue sections demonstrated that the antigen was localized predominantly to the surface membrane of lymphocytes, and that canine lymphoma cells expressed a greater amount of the antigen than normal lymph node lymphocytes. It was concluded that these structurally and antigenically related high molecular weight proteins, based on their antigenic patterns, limited peptide analysis and tissue distribution, represent the canine homologue of the lymphocyte differentiation antigen known as T200.