Complete deletion of Apc results in severe polyposis in mice.

The adenomatous polyposis coli (APC) gene product is mutated in the vast majority of human colorectal cancers. APC negatively regulates the WNT pathway by aiding in the degradation of beta-catenin, which is the transcription factor activated downstream of WNT signaling. APC mutations result in beta-catenin stabilization and constitutive WNT pathway activation, leading to aberrant cellular proliferation. APC mutations associated with colorectal cancer commonly fall in a region of the gene termed the mutation cluster region and result in expression of an N-terminal fragment of the APC protein. Biochemical and molecular studies have revealed localization of APC/Apc to different sub-cellular compartments and various proteins outside of the WNT pathway that associate with truncated APC/Apc. These observations and genotype-phenotype correlations have led to the suggestion that truncated APC bears neomorphic and/or dominant-negative function that support tumor development. To analyze this possibility, we have generated a novel allele of Apc in the mouse that yields complete loss of Apc protein. Our studies reveal that whole-gene deletion of Apc results in more rapid tumor development than the APC multiple intestinal neoplasia (Apc(Min)) truncation. Furthermore, we found that adenomas bearing truncated Apc had increased beta-catenin activity when compared with tumors lacking Apc protein, which could lead to context-dependent inhibition of tumorigenesis.

In vitro culture of coxsackievirus group B, type 3 immune spleen cells on infected endothelial cells and biological activity of the cultured cells in vivo.

Spleen cells from male BALB/c mice infected 7 days earlier by an intraperitoneal injection of 3 X 10(4) PFU of a myocarditic strain of coxsackievirus B-3 lysed virus-infected endothelial cells in a 51Cr release assay. Cytotoxic activity in the in vivo sensitized spleen cell population could be further increased by culturing the immune spleen cells from infected mice on virus-infected or uninfected endothelial cells for 6 to 7 days in vitro. Cytotoxicity of in vitro cultured spleen cells to infected targets was mediated by T lymphocytes since reactivity was abolished by treatment of the spleen cells with anti-thy 1.2 serum and complement. Reciprocal assays with BALB/c and C57BL cells indicated that maximum cytotoxicity occurred when spleen cells were sensitized on syngeneic endothelial cells. Other experiments showed that spleen cells sensitized to coxsackievirus B-3 or encephalomyocarditis virus were selectively cytolytic to targets infected with the homologous virus. Adoptive transfer of T cells cultured in vitro on infected endothelial cells retained their ability to induce myocarditis in T-lymphocyte-deficient mice.

Sex-related differences in the pattern of coxsackievirus B-3-induced immune spleen cell cytotoxicity against virus-infected myofibers.

Spleen cells from adult BALB/c mice injected intraperitoneally with purified coxsackievirus B-3 were tested for cytotoxicity against 51Cr-labeled syngeneic infected and uninfected myofibers. Both male and female immune cells were active against uninfected targets; this reactivity was evident by day 3 of infection and persisted throughout the first week. However, we observed marked sex-related differences in immune cell cytotoxicities against infected myofibers. Males exhibited a strong T-lymphocyte response 4 to 7 days after infection. In contrast, females exhibited a weak response, and only infrequently were the immune spleen cells of females significantly more reactive against infected myofibers than against uninfected myofibers. The demonstration of a stronger effector cell response against infected myocardial cells in male mice correlates with the observation that clinical adult coxsackievirus B myocarditis and pericarditis occur predominantly in males.

Sex-related differences in the rapid production of cytotoxic spleen cells active against uninfected myofibers during Coxsackievirus B-3 infection.

Spleen cells from BALB/c mice immunized with a cardiotropic strain of Coxsackie B-3 virus were more cytolytic to uninfected myofibers than were spleen cells from mice immune to a noncardiotropic strain of the virus. Spleen cells immune to either virus were equally cytolytic to endothelial cells. Cytotoxicity was greater in female mice than in males. Analysis of individual reactivities showed that the male response was heterogeneous with only half of the animals developing cytolytic activity. All females responded. Early (day 3) cytolytic cells in both male and female mice appear to be natural killer (NK) cells, since they are not sensitive to anti-thy 1.2 or anti-Ig serum and complement, and lyse allogeneic (CBA) as well as syngeneic (BALB/c) targets. Later (days 4 to 6), the cytolytic cells in males become sensitive to anti-thy 1.2 serum and are restricted to lysis of syngeneic targets, while the cytolytic cells in females maintain the characteristics of NK cells.

Lysis of infected myofibers by coxsackievirus B-3-immune T lymphocytes.

Spleen cells from male adult BALB/c mice given intraperitoneal injections of purified coxsackievirus B-3 were examined for the ability to lyse syngeneic neonatal myofibers in culture. Cytotoxicity against infected and uninfected targets was measured with the use of an in vitro 51Cr release assay. Immune spleen cells obtained 4--7 days after infection were cytotoxic for viral-infected myofibers. Peak reactivity was observed 5 days after infection. At this time immune spleen cells showed significantly less reactivity against uninfected myofibers. Cytotoxicity against infected targets was mediated by T lymphocytes, since reactivity was abolished by treatment with anti-thy 1.2 and complement. Treatment with anti-Ig and complement caused no loss of activity. Reciprocal assays performed with BALB/c and CBA cells showed that maximal cytotoxicity occurred against infected syngeneic myofibers, providing further evidence that viral-specific effector cells were T lymphocytes. In addition, hyperimmune rabbit anti-coxsackievirus B-3 antiserum could not block immune spleen cell lysis of infected targets, suggesting that coxsackievirus-infected myofibers expressed surface membrane antigens not recognized by specific neutralizing antibody.

Lack of correlation between neutralizing antibody production and suppression of coxsackievirus B-3 replication in target organs: evidence for involvement of mononuclear inflammatory cells in host defense.

CD-1 mice inoculated with coxsackievirus B-3 i.p. developed a generalized infection involving the heart, pancreas, and liver. The disease was nonlethal and viral growth in the target organs was terminated in about a week. Administration of cortisone acetate 30 min to 2 hr before infection markedly enhanced the severity of disesae. Abnormally high titers of virus were found in the target organs between days 3 and 7 with persistence of infectious virus in the heart for at least 2 weeks. In addition the extent of necrosis of myofibers, pancreatic acini, and hepatic parenchyma was increased and a high percentage of the animals died. There was no evidence that the anti-viral antibody response was impaired in steroid-treated mice since concentrations of neutralizing antibody in the circulation were normal. In contrast, immigration of mononuclear inflammatory cells into the hearts of these animals was depressed and when present, foci of inflammation contained some polymorphonuclear leukocytes. The data indicate that inhibition of coxsackieviral growth cannot be attributed to the sole effects of neutralizing antibody and suggest that mononuclear inflammatory cells infiltrating the heart play a role in primary host defense.

Generation of cytotoxic T lymphocytes during coxsackievirus B-3 infection. I. Model and viral specificity1.

The production of cytotoxic cells in the spleen of adult male BALB/c mice infected with Coxsackievirus B-3 has been examined. An in vitro 51Cr release assay was used to measure cytotoxic activity against virus-infected and uninfected neonatal sygeneic fibroblasts. Cytotoxicity of immune spleen cells against virus-infected targets was detected on the 3rd day after infection, reached a peak on day 7, and then declined to low levels by days 12 and 14. Spleen cells obtained 3 and 5 days after infection also exerted cytotoxicity against uninfected fibroblasts, but by the 7th day there was little or no reactivity against uninfected target cells, although activity against infected fibroblasts was maximal at this time. Reciprocal assays performed by using Coxsackie and vaccinia viruses provided evidence of virus specificity of the cytotoxic reaction. When spleen cells were obtained 7 days after infection, the Coxsackievirus-immune population was not cytotoxic for vaccinia-infected fibroblasts, and the vaccinia-immune population was not cytotoxic for Coxsackievirus-infected targets, although each immune cell preparation caused significant lysis of fibroblasts infected with the homologous virus. Additional studies showed that primary mouse or hyperimmune rabbit anti-Coxsackieviral serum could not block immune spleen cell cytotoxicity or induce complement-mediated lysis of infected targets. The findings indicate that Coxsackievirus infection results in surface membrane alterations, but no evidence was obtained that antiviral antibody could react with the infected cells.

Generation of cytotoxic T lymphocytes during coxsackievirus tb-3 infection. II. Characterization of effector cells and demonstration cytotoxicity against viral-infected myofibers1.

This report describes studies characterizing the virus-specific cytotoxic effector cells which are present in the spleens of mice 7 days after infection with Coxsackievirus B-3. An in vitro 51Cr assay employing eyngeneic virus-infected neonatal fibroblasts was used to measure cytotoxic activity. Treatment of immune cells with (anti-thy-1.2) and complement abolished dtheir cytotoxic activity, but no reduction occurred when B cells were removed by incubation with anti-Ig and complement or macrophages eliminated by adherence depletion. The findings therefore imply that the cytotoxic reaction was mediated by sensitized T cells and that B cells and macrophages did not play an important role. Reciprocal assays performed with BALB/c and CBA/J cells showed that Coxsackievirus-immune spleen cells lysed infected syngeneic targets but not allogeneic targets, providing further evidence that cytotoxicity was mediated by effector T cells. In addition and in vitro assay system employing neonatal myocardial cells was developed and used to demonstrate that Coxsackievirus-infected myofibers were susceptible to destruction by immune spleen cells. The evidence suggests that mice infected with Coxsackie B viruses are able to mount a cell-mediated immune response with production of cytotoxic T cells which have the capacity to damage tissues infected with these agents.

Influenza A virus interaction with murine lymphocytes. I. The influence of influenza virus A/Japan 305 (H2N2) on the pattern of migration of recirculating lymphocytes.

The effect of influenza virus A/Japan 305 (H2N2) on the path of migration of recirculating lymphocytes has been studied. 51Cr-labeled rat thoracic duct lymphocytes (TDL) were incubated with virus at 37 degrees C for 1 hr and then infused i.v. into syngeneic recipients which were killed 1 hr later. Virus-treated TDL accumulated in the liver and their recovery in lymph nodes and spleen was severely reduced. Changes in lymphocytes induced by virus developed rapidly and were evident after incubation for only 15 min. UV-irradiated virus altered the pattern of lymphocyte localization but attachment of heat-inactivated virus to lymphocytes in vitro had no effect on their distribution in vivo. Evidence was obtained that some virus-treated TDL, initially sequestered in the liver, subsequently recovered their ability to circulate normally. Recovery was not complete and a population of cells failed to regain their ability to home into lymph nodes. Evidence is also presented demonstrating that influenza virus affected the homing properties of both T and B cells. It is suggested that aberrations in lymphocyte homing were mediated by the viral neuraminidase which induces changes in the cell membrane leading to their accumulation in the liver.

Influenza A virus interaction with murine lymphocytes. II. Changes in lymphocyte surface properties induced by influenza virus A/Japan 305 (H2N2).

This report describes changes in surface properties of rat thoracic duct lymphocytes (TDL) induced by influenza virus A/Japan 305 (H2N2). After incubation with virus at 37 degrees C, sialic acids were released from the membranes of TDL WITH CONCOMItant reduction in their mean electrophoretic mobility. Autoradiographs of TDL which had been incubated with 125I virus at 4 degrees C showed that the particles attached to nearly all the lymphocytes although there was variation in the amount of virus bound per cell. Attachment occurred rapidly with maximum labeling evident after incubation at 4 degrees C for 5 min. Incubation at 37 degrees C for 1 hr resulted in a 10 to 18% reduction in the incidence of labeled TDL suggesting that some virus eluted spontaneously. When 125I-virus-treated TDL were incubated in syngeneic serum, substantial amounts of virus eluted reducing the incidence of labeled cells by 50%. In addition, evidence was obtained by indirect immunofluorescence that syngeneic serum contains antibodies which react with sites on the lymphocyte surface exposed by the viral neuraminidase.

Prolonged allograft survival in Newcastle disease virus-treated mice.

There was persistent lymphocytopenia and prolonged survival of C57Bl skin allografts when recipient BALB/c mice were injected daily with Newcastle disease virus.