Neonatal death in mice lacking cardiotrophin-like cytokine is associated with multifocal neuronal hypoplasia.

Mice with null mutations of ciliary neurotrophic factor (Cntf) receptor alpha (Cntf-Ralpha), or cytokine-like factor 1 (Clf), one component of Cntf-II (a heterodimeric Cntf-Ralpha ligand), die as neonates from motor neuron loss affecting the facial nucleus and ventral horn of the lumbar spinal cord. Exposure to cardiotrophin-like cytokine (Clc), the other putative Cntf-II element, supports motor neuron survival in vitro and in ovo. Confirmation that Clc ablation induces an equivalent phenotype to Clf deletion would support a role for Clc in the functional Cntf-II complex. In this study, Clc knockout mice had decreased facial motility, did not suckle, died within 24 hours, and had 32% and 29% fewer motor neurons in the facial nucleus and lumbar ventral horn, respectively; thus, Clc is essential for motor neuron survival during development. The concordance of the Clc knockout phenotype with those of mice lacking Cntf-Ralpha or Clf bolsters the hypothesis that Clc participates in Cntf-II.

Repair-deficient 3-methyladenine DNA glycosylase homozygous mutant mouse cells have increased sensitivity to alkylation-induced chromosome damage and cell killing.

In Escherichia coli, the repair of 3-methyladenine (3MeA) DNA lesions prevents alkylation-induced cell death because unrepaired 3MeA blocks DNA replication. Whether this lesion is cytotoxic to mammalian cells has been difficult to establish in the absence of 3MeA repair-deficient cell lines. We previously isolated and characterized a mouse 3MeA DNA glycosylase cDNA (Aag) that provides resistance to killing by alkylating agents in E. coli. To determine the in vivo role of Aag, we cloned a large fragment of the Aag gene and used it to create Aag-deficient mouse cells by targeted homologous recombination. Aag null cells have no detectable Aag transcripts or 3MeA DNA glycosylase activity. The loss of Aag renders cells significantly more sensitive to methyl methanesulfonate-induced chromosome damage, and to cell killing induced by two methylating agents, one of which produces almost exclusively 3MeAs. Aag null embryonic stem cells become sensitive to two cancer chemotherapeutic alkylating agents, namely 1,3-bis(2-chloroethyl)-1-nitrosourea and mitomycin C, indicating that Aag status is an important determinant of cellular resistance to these agents. We conclude that this mammalian 3MeA DNA glycosylase plays a pivotal role in preventing alkylation-induced chromosome damage and cytotoxicity.

Immune function in mice lacking the perforin gene.

Mice lacking the perforin gene were generated by using targeted gene disruption in embryonal stem cells. When infected with lymphocytic choriomeningitis virus (LCMV), perforin-less (-/-) mice showed clear signs of having mounted an immune response based on activation of CD8 T cells but were unable to clear the LCMV infection. This failure to eliminate virus was accompanied by a failure to generate spleen cells capable of lysing LCMV-infected fibroblasts in vitro. Spleen cells from LCMV-infected -/- mice were able to lyse hematopoietic target cells after exposure to phorbol 12-myristate 13-acetate and ionomycin, provided the target cells expressed the Fas antigen. Spleen cells from -/- mice also responded to alloantigen in mixed leukocyte culture by blastogenesis and proliferation. The resulting cells were able to lyse hematopoietic target cells, although not as well as spleen cells from +/+ littermates sensitized in the same manner. However, lysis by -/- cells was again seen only if the target cells expressed Fas antigen. We conclude that perforin-less -/- mice retain and express the Fas lytic pathway as expressed in vitro but that this pathway is insufficient to clear an LCMV infection in vivo.

Abnormal T cell development in CD3-zeta-/- mutant mice and identification of a novel T cell population in the intestine.

The T cell antigen receptor (TCR)-associated invariable membrane proteins (CD3-gamma, -delta, -epsilon and -zeta) are critical to the assembly and cell surface expression of the TCR/CD3 complex and to signal transduction upon engagement of TCR with antigen. Disruption of the CD3-zeta gene by homologous recombination resulted in a structurally abnormal thymus which primarily contained CD4- CD8- and TCR/CD3very lowCD4+CD8+ cells. Spleen and lymph nodes of CD3-zeta-/- mutant mice contained a normal number and ratio of CD4+ and CD8+ single positive cells that were TCR/CD3very low. These splenocytes did not respond to antibody cross-linking or mitogenic triggering. The V beta genes of CD4-CD8- and CD4+CD8+ thymocytes and splenic T cells were productively rearranged. These data demonstrated that (i) in the absence of the CD3-zeta chain, the CD4- CD8- thymocytes could differentiate to CD4+CD8+ TCR/CD3very low thymocytes, (ii) that thymic selection might have occurred, (iii) but that the transition to CD4+CD8- and CD4-CD8+ cells took place at a very low rate. Most strikingly, intraepithelial lymphocytes (IELs) isolated from the small intestine or the colon expressed normal levels of TCR/CD3 complexes on their surface which contained Fc epsilon RI gamma homodimers. In contrast to CD3-zeta containing IELs, these cells failed to proliferate after triggering with antibody cross-linking or mitogen. In comparison to thymus-derived peripheral T cells in the spleen and lymph nodes, the preferential expression of normal levels of TCR/CD3 in intestinal IELs suggested they mature via an independent extrathymic pathway.

Immunoglobulin gene rearrangement in B cell deficient mice generated by targeted deletion of the JH locus.

B lymphocyte differentiation is characterized by an ordered series of Ig gene assembly and expression events. In the majority of normal B cells, assembly and expression of Ig heavy (H) chain genes precedes that of light (L) chain genes. To determine the role of the Ig heavy chain protein in B cell development and L chain gene rearrangement, we have generated mice that cannot assemble Ig H chain genes as a result of targeted deletion of the JH gene segments in embryonic stem cells. Mice homozygous for this deletion are devoid of slg+ B cells in the bone marrow and periphery. B cell differentiation in these mice is blocked at the large, CD43+ precursor stage. However, these precursor B cells do assemble kappa L chain genes at a low level in the absence of mu H chain proteins. These data demonstrate that rearrangement and expression of the mu H chain gene is not absolutely required for kappa L chain gene rearrangement in vivo. Expression of mu chains may facilitate either efficient L chain gene rearrangement or the survival of cells that have rearranged light chain genes by promoting the differentiation of large, CD43+ to small, CD43- pre-B cells.

Transgenic mice containing a human heavy chain immunoglobulin gene fragment cloned in a yeast artificial chromosome.

We have developed a method for the introduction of yeast artificial chromosomes (YACs) into transgenic mice. An 85 kilobase (kb) fragment of the human heavy chain immunoglobulin gene was cloned as a YAC, and embryonic stem cell lines carrying intact, integrated YACs were derived by co-lipofection of the YAC with an unlinked selectable marker. Chimaeric founder animals were produced by blastocyst injection, and offspring transgenic for the YAC were obtained. Analysis of serum from these offspring for human heavy chain antibody subunits demonstrated expression of the YAC-borne immunoglobulin gene fragment. Co-lipofection may prove to be a highly-successful means of producing transgenic mice containing large gene fragments in YACs.

B cell development in mice that lack one or both immunoglobulin kappa light chain genes.

We have generated mice that lack the ability to produce immunoglobulin (Ig) kappa light chains by targeted deletion of J kappa and C kappa gene segments and the intervening sequences in mouse embryonic stem cells. In wild type mice, approximately 95% of B cells express kappa light chains and only approximately 5% express lambda light chains. Mice heterozygous for the J kappa C kappa deletion have approximately 2-fold more lambda+ B cells than wild-type littermates. Compared with normal mice, homozygous mutants for the J kappa C kappa deletion have about half the number of B cells in both the newly generated and the peripheral B cell compartments, and all of these B cells express lambda light chains in their Ig. Therefore, homozygous mutant mice appear to produce lambda-expressing cells at nearly 10 times the rate observed in normal mice. These findings demonstrate that kappa gene assembly and/or expression is not a prerequisite for lambda gene assembly and expression. Furthermore, there is no detectable rearrangement of 3' kappa RS sequences in lambda+ B cells of the homozygous mutant mice, thus rearrangements of these sequences, per se, is not required for lambda light chain gene assembly. We discuss these findings in the context of their implications for the control of Ig light chain gene rearrangement and potential applications of the mutant animals.

Prevention of doxorubicin-induced hematotoxicity in mice by interleukin 1.

Interleukin 1 alpha and interleukin 1 beta induce peripheral neutrophilia with stimulation of granulopoiesis in bone marrow. The continuous administration of interleukin 1 (100 ng/day) to mice for 7 days by s.c.-implanted Alzet osmotic minipumps induced marked stimulation of granulopoiesis in marrow and spleen in normal mice, and protected against the marked depletion of myeloid and erythroid cells in bone marrow of mice treated with single injections of either 20 or 30 mg/kg doxorubicin (DXN). Interleukin 1 beta infusion also protected against DXN-induced atrophy of thymus and secondary lymphoid organs. Single i.p. injection of either interleukin 1 alpha or interleukin 1 beta at doses up to 1000 ng 24 h prior to treatment with DXN did not protect against the hematopoietic and lymphoid toxicities of DXN.

Enhanced antitumor efficacy of adriamycin in combination with interferon-gamma: effects on macrophages.

The combination of the immunomodulator interferon-gamma (IFN-gamma) with the chemotherapeutic drug adriamycin (ADM) was assessed in vitro and in vivo in murine tumor models. When tested in vivo against the murine Lewis lung carcinoma, significantly greater reduction of spontaneous pulmonary metastases was obtained by combination treatment with IFN-gamma, followed 1 day later by ADM. Intraperitoneal ADM treatment also resulted in an increased recruitment of peritoneal mononuclear cells. It is noteworthy that, although the antitumor efficacy was significantly increased by the IFN-gamma/ADM combination treatment, gross toxicity of ADM was not increased. Thus, a net increase in the therapeutic index of ADM was achieved. In vitro, the effects of ADM on the ability of murine peritoneal macrophages, with or without the addition of immunological macrophage activators, to kill tumor cells was studied. Resident macrophages were able to sequester ADM (when present at 10 micrograms/ml) from the medium, and could subsequently mediate killing of target tumor cells. However, incubation of macrophages with low (ineffective by themselves) doses of ADM (1 microgram/ml) prevented their simultaneous or subsequent activation to the tumoricidal state after incubation with the normal macrophage-activating mixture of IFN-gamma plus a muramyl dipeptide (MDP) analog. When the order of addition of reagents was reversed such that the macrophages were preincubated for 24 hr with IFN-gamma (100 U/ml) plus the MDP analog (0.1-10 micrograms/ml), no antagonism of tumoricidal activity was obtained upon subsequent incubation with ADM. There were no interactions between IFN-gamma and ADM on the direct proliferation of tumor cells. Taken together, these results suggest that the enhanced antitumor efficacy of IFN-gamma/ADM combinations in vivo was not due to direct antiproliferative effects on the tumor cells, but rather may be mediated by direct cytotoxicity of ADM on tumor cells enhanced by phagocytic mononuclear cells.

Prophylaxis and treatment of experimental lung metastases in mice after treatment with liposome-encapsulated 6-O-stearoyl-N-acetylmuramyl-L-alpha-aminobutyryl-D-isoglutamine.

A new lipophilic muramyl dipeptide analog, 6-O-stearoyl-N-acetylmuramyl-L-alpha-aminobutyryl-D-isoglutamine, when incorporated in liposomes, was effective in both the prevention and eradication of experimental pulmonary metastases in mice. Multilamellar vesicles composed of synthetic phospholipids (phosphatidylglycerol and phosphatidylcholine) containing saturated myristoyl or unsaturated dioleoyl acyl chains were found to potentiate the antimetastatic activity of this glycopeptide. Prophylactic and therapeutic efficacy was observed against the three murine tumors tested: FSa, an immunogenic fibrosarcoma; NFSa, a nonimmunogenic fibrosarcoma; and B16 melanoma. Neither the administration of empty liposomes or free glycopeptide, nor their coadministration, had a significant antimetastatic effect. This approach is promising for the therapy of cancer metastases in humans, particularly in the prevention of metastatic seeding and in the treatment of micrometastases.

A defect in cell-mediated immune function in insulin-resistant diabetic and obese subjects.

In this study, we have evaluated one aspect of the cellular immune process--in vitro release of MIF by lymphocytes obtained from controls; well-controlled, ketosis-prone, insulin-dependent diabetic patients; nonketotic diabetic patients; and nonhyperglycemic obese patients. The results showed that MIF release by cells from well-controlled, insulin-dependent diabetic, nonketotic diabetic, and nonhyperglycemic obese subjects was 100% +/- 8, 48% +/- 17, and 36% +/- 17 of control values, respectively. Thus insulin-sensitive, ketosis-prone diabetic patients have normal MIf release while well-controlled on insulin therapy, whereas insulin-resistant, nonketotic diabetic and nonhyperglycemic obese patients have impaired MIF release. It is possible that this in vitro defect in a cellular immune process is related to the insulin-resistant state and that it may play a role in enhanced susceptibility to infection in insulin-resistant diabetic patients.

The effect of variations in vitamin C intake on the cellular immune response of guinea pigs.

The uptake of tritiated thymidine by isolated peripheral blood lymphocytes obtained from male guinea pigs immunized with bovine serum albumin was studied in animals maintained on various amounts of Vitamin C for 28 days. Animals were pair-fed on ascorbate-free diet and were supplemented intraperitoneally with 0, 25, or 250 mg Na-ascorbate per day. Scorbutic animals lost weight rapidly during the final 2 experimental weeks. Their daily food intake averaged only 4 g/day during the last week; thus, pair-fed ascorbate-supplemented groups were also subjected to acute nutritional stress. Lymphocytes from guinea pigs receiving 250 mg Na-ascorbate per day incorporated in vitro the highest amounts of tritiated thymidine both in the absence of nonspecific mitogen and in the presence of concanavalin A or phytohemagglutinin. Responses to lipopolysaccharide were not conclusive. Total circulating white cells counts and relative numbers of T and B lymphocytes were assessed in a second study made under identical constraints. In scorbutic animals the percentage of B lymphocytes increased and that of T lymphocytes decreased continuously over the 4-week period. The opposite effect was observed in vitamin C-supplemented animals. These studies suggest that very high doses of ascorbate support elevated mitotic activity after 4 weeks of much reduced food intake.

Cell-mediated cytotoxicity and humoral immune response in ascorbic acid-deficient guinea pigs.

Guinea pigs were divided into three dietary groups: ascorbic-acid deficient, pair-fed, and ad libitum control. Two weeks later guinea pigs were immunized intradermally with 5 x 10(8) chicken erythrocytes in Freund's complete adjuvant. Hemagglutinating antibody titers to chicken erythrocytes 2 weeks after immunization were comparable in all three dietary groups. In vitro 51Cr release from labeled chicken erythrocyte target cells incubated with lymphoid cells from spleens of ascorbic acid-deficient guinea pigs was significantly less than with spleen cells from pair-fed and ad libitum control guinea pigs. The percentage of splenic lymphoid cells that formed rosettes with rabbit erythrocytes, a T cell marker, was the same in all three dietary groups. The defect of ascorbic acid deficiency may reflect an impairment of T lymphocytes function in cell-mediated cytotoxicity or a change in number or function of another cell type.

Rauscher virus-induced reticulum-cell sarcomas: Their growth in vitro and erythropoietic differentiation.

A transplantable reticulum-cell sarcoma induced by Rauscher virus (RV) in (C57BL/6 X DBA/2)F1 (BDF1) mice was grown in tissue culture. Four separate cell lines were established, all of which grew predominantly in suspension. The doubling time of the cells from these cultures ranged from 17 to 32 h. Each culture continued to replicate RV, as indicated by the infectivity in newborn mice of all fluids tested up to the 75th passage. Since the morphological appearance of the cells in vitro was consistent with that of proerythroblasts, all cultures were tested for their ability to differentiate along the erythrocytic line under the influence of dimethylsulphoxide (DMSO). One of the cultures produced small quantities of haemoglobin independently of DMSO. Another was shown to produce haemoglobin, as well as to take up 59Fe and incorporate it into haem, only in the presence of DMSO. The 2 remaining cultures failed to produce haemoglobin, either spontaneously or in the presence of DMSO. Cells from each of the RV-induced cultures, when inoculated back into BDF1 mice, induced typical reticulum-cell sarcomas, without in vivo evidence of erythroid differentiation. In contrast, 2 morphologically identical but non-infectious cell lines derived from Friend virus-induced reticulum-cell sarcomas did not show erythroid differentiation in vivo or in vitro, either in the absence or presence of DMSO.

Helper specificity for retrieval of defective friend virus.

A Friend virus-induced reticulum-cell sarcoma from BALB/c mice has been cultivated in vitro in our laboratory for more than 11 years and contains no detectable evidence of virus. However, Friend virus could be retrieved readily from the tissue cultures by any of several lymphatic leukemia viruses belonging to the Friend-Moloney-Rauscher (FMR) group, as long as the helper viruses were actively replicating in a culture. Helper activity appeared to be highly specific and limited to the FMR group including the B-tropic Tennant leukemia virus which produced a B-tropic Friend virus pseudotype. The naturally occurring type AKR murine leukemia viruses and the related Gross passage A virus failed both in vivo and in vitro to retrieve the Friend virus genome from the virus-free tumor cells.