Toxicologic evaluation of injectable acemannan in the mouse, rat and dog.

Acemannan, the USAN-accepted name for long-chain polydispersed beta-(1,4)-acetylated polymannose with interspersed 0-acetyl groups with a mannose monomer/acetyl ratio of approximately 1:1 and extracted from Aloe vera (barbadensis Miller), was administered as a 1.0 mg/ml solution to mice, rats and dogs, either as single dose or repeated at 4-d intervals for 8 doses by iv or ip routes. No significant signs of intoxication and no deaths occurred in animals treated with the single injection of acemannan at dosages of 80 mg/kg iv or 200 mg/kg ip in mice, 15 mg/kg iv or 50 mg/kg ip in rats, and 10 mg/kg iv or 50 mg/kg ip in dogs. On repeated injections systemic toxicity was limited to obvious transient discomfort that appeared dose related. There was accumulation of macrophages and monocytes without subsequent inflammatory reaction in lungs of the iv-treated animals, and in liver and spleen and on peritoneal surfaces of ip-treated animals. The effects were not considered adverse, but were consistent with the known immune stimulating activity of acemannan. A few deaths occurred in mice and rats that were suggestive of resulting from improper injection or sequella of necrosis of the injection site. The NOAELs for acemannan determined from these repeated injection studies were 20 mg/kg iv or ip in the mouse, 4.0 mg/kg iv and 50 mg/kg ip in the rat, and 1.0 mg/kg iv in dogs; 5.0 mg acemannan/kg ip in the dog was considered to be LOAEL, based on the emesis and abdominal discomfort induced.

Pristane induced arthritis. II. Genetic regulation in F1 hybrid mice and cellular immune abnormalities following pristane injection.

Pristane-induced arthritis was investigated in DBA/1, DBA/2, and BALB/c mice, and F1 hybrid mice generated from inter-crosses between these strains. The incidence of disease in F1 hybrid mice was significantly lower than the susceptible parental strains (DBA/1 and BALB/c), and resistance to arthritis was observed in both DBA/2 mice and the (DBA/2 x BALB/c) F1 hybrid mice. Several cellular immune abnormalities were observed in pristane-injected DBA/1 mice. Con A mitogen responses were depressed following pristane injection, and a functional suppressor cell population was detected. Delayed type hypersensitivity responses to type II collagen were observed in pristane injected mice. The intraperitoneal injection of pristane appears to alter immune regulation and induce autoimmune responses to connective tissue components.

Collagen-induced arthritis in mice. VI. Synovial cells from collagen arthritic mice activate autologous lymphocytes in vitro.

Synovial cells were extracted from normal and collagen-arthritic mice and investigated for lymphocyte-activating properties. In mixed cell culture, irradiated fibroblast-like synovial cells from DBA/1 LacJ arthritic mice stimulated a strong proliferative response in spleen cells from syngeneic normal mice, but not in cells from allogeneic DBA/2. B10.RIII, or BALB/c mice. This novel stimulus occurred in the absence of detectable Class II MHC antigen expression on the fibroblast-like synovial cell surface or increased autologous mixed lymphocyte reactions between DBA/1 LacJ spleen and lymph node cells. Irradiated synovial cells were also unable to present type II collagen to a collagen-specific T cell line and to stimulate proliferation. Addition of interferon-gamma or interleukin-1 failed to induce detectable surface Ia on the synovial fibroblasts or induce the capacity for antigen presentation in these cells.

Type II collagen induced arthritis in mice. V. The role of the spleen cell response in the immune and arthritogenic reaction to type II collagen.

The contribution of the spleen to the arthritogenic and immune response to type II collagen in mice was measured by a series of in vitro and in vivo experiments. Negligible antibody production and proliferative responses to collagen were measured in the spleen from immunized mice, compared to lymph node and peripheral blood. Further, splenectomized mice were found to be susceptible to collagen induced arthritis with a similar disease incidence to sham operated controls. There were no major differences in the sera antibody responses or the delayed-type hypersensitivity responses to collagen between the splenectomized and control mice. The primary regulation of the response to type II collagen in collagen induced arthritis was apparently independent of the spleen cell population.

Pristane-induced arthritis. The immunologic and genetic features of an experimental murine model of autoimmune disease.

Pristane was injected intraperitoneally into mice of several strains, inducing an inflammatory seropositive arthritis in susceptible strains. The evolving histologic features included synovial hyperplasia, periostitis, and progressive marginal erosions. Multiple serologic immune abnormalities, including rheumatoid factor and anticollagen antibodies, also developed. Genetic analysis indicated that the major histocompatibility complex (H-2), C5 hemolytic complement (Hc), Newcastle disease virus-induced interferon (IF-1), and athymic (nu/nu) loci were involved in regulating susceptibility to pristane-induce arthritis. This experimental murine disease may provide a novel model of rheumatoid arthritis.

T cells discriminate between Ia antigens expressed on allogeneic accessory cells and B cells: a potential function for carbohydrate side chains on Ia molecules.

Previous studies have shown that the peptides obtained from accessory cell and B-cell Ia molecules are identical but that the alpha chains of B-cell Ia molecules are more extensively sialylated than those of accessory cells. The present studies were designed to determine whether this glycosylation difference can account for the functional difference in the capacity of the two cell types to activate alloreactive T cells. The experimental data show that normal resting B cells lack the capacity to induce DNA synthesis or differentiation in alloreactive T cells. T cells do recognize polymorphisms in B-cell Ia molecules, however, because they can be specifically primed for a subsequent proliferative stimulus of the same haplotype. The mitogenic signal for T cells is delivered by either allogeneic accessory cells or neuraminidase-treated B cells. Therefore, the T-cell receptor(s) may contain a site specific for the nonpolymorphic asialocarbohydrate moiety on the alpha chains of accessory cell Ia molecules.

Use of hybridoma-resident variant cell lines to study H-2Db/FMR-specific cytotoxic T cells.

An H-2Db heterozygous tumor cell line and a variant subclone bearing a mutant gene product were used to analyze the H-2Db specificity of cytotoxic T lymphocytes (CTL) generated during a Moloney murine sarcoma virus (MSV) infection. When the mutant cells were used as targets for MSV-specific CTL, the amount of cell lysis, compared with that seen with the nonmutant parental cells, was drastically decreased. However, cells of the mutant clones remained susceptible to allogeneic CTL specific for the nonmutant H-2Db molecule. The mutant cells also did not differ from the parent cells in their level of viral antigen expression. Biochemically the parental and mutant molecules were similar but not identical. The data indicate that minor alterations of the H-2 antigens caused by somatic mutation may prevent virus-infected cells from being recognized as targets by CTL.

Tumors induced by murine sarcoma virus contain precursor cells capable of generating tumor-specific cytolytic T lymphocytes.

Leukocyte fractions extracted from the tumor mass and the lymphoid organs of C57BL/6 (B6) mice carrying murine sarcoma virus-induced tumors contained primed cytolytic T-lymphocyte (CTL) precursor cells, in addition to active cytotoxic T cells. These leukocyte fractions gave a secondary response when stimulated in vitro with syngeneic tumor cells, generating large numbers of specific CTL. The activity of these CTL (H-2b) was apparently H-2-restricted, because it was ineffective on tumor targets bearing strongly cross-reacting tumor-specific antigens but with the H-2d haplotype. Furthermore, only H-2b cells bearing the Friend, Moloney, Rauscher-associated antigen, such as Rauscher leukemia virus-induced RBL-5 cells and Friend leukemia virus-induced HFL/b cells, were lysed efficiently. B male GV cells (H-2b cells induced by Gross leukemia virus) were not affected by the same CTL. We propose the existence of a dynamic state involving the migration of primed CTL precursor cells between the lymphoid organs and the tumor mass, as well as the differentiation of these precursor cells within the tumor mass into highly specific CTL.