Unchallenged Mice Research Articles

Linked deficiencies in extracellular PP(i) and osteopontin mediate pathologic calcification associated with defective PC-1 and ANK expression.

Osteopontin and PP(i) both suppress hydroxyapatite deposition. Extracellular PP(i) deficiency causes spontaneous hypercalcification, yet unchallenged osteopontin knockout mice have only subtle mineralization abnormalities. We report that extracellular PP(i) deficiency promotes osteopontin deficiency and correction of osteopontin deficiency prevents hypercalcification, suggesting synergistic inhibition of hydroxyapatite deposition. Nucleotide pyrophosphatase phosphodiesterase (NPP) isozymes including PC-1 (NPP1) function partly to generate PP(i), a physiologic calcification inhibitor. PP(i) transport is modulated by the membrane channel protein ANK. Spontaneous articular cartilage calcification, increased vertebral cortical bone formation, and peripheral joint and intervertebral ossific ankylosis are associated with both PC-1 deficiency and expression of truncated ANK in ank/ank mice. To assess how PC-1, ANK, and PP(i) regulate both calcification and cell differentiation, we studied cultured PC-1 -/- and ank/ank mouse calvarial osteoblasts. PC-1 -/- osteoblasts demonstrated approximately 50% depressed NPP activity and markedly lowered extracellular PP(i) associated with hypercalcification. These abnormalities were rescued by transfection of PC-1 but not of the NPP isozyme B10/NPP3. PC-1 -/- and ank/ank cultured osteoblasts demonstrated not only comparable extracellular PP(i) depression and hypercalcification but also marked reduction in expression of osteopontin (OPN), another direct calcification inhibitor. Soluble PC-1 (which corrected extracellular PP(i) and OPN), and OPN itself (> or = 15 pg/ml), corrected hypercalcification by PC-1 -/- and ank/ank osteoblasts. Thus, linked regulatory effects on extracellular PP(i) and OPN expression mediate the ability of PC-1 and ANK to regulate calcification.

Effect of a single dose of ponazuril on neural infection and clinical disease in Sarcocystis neurona-challenged interferon-gamma knockout mice

Interferon γ-knockout mice were challenged with 5000 Sarcocystis neurona sporocysts acquired from a naturally infected opossum. Ponazuril was administered once, by gavage, at day 1, 3, 7, 10, or 14 post-infection (pi). Ponazuril was given at either 20 or 200 mg/kg. Mice that survived to day 30 pi were euthanized. Severity of CNS infection was quantified as schizont density in the cerebellum. Unchallenged mice in treatment and non-treatment groups remained free of disease and gained weight throughout the experiment. All challenged mice, regardless of treatment, developed histologic evidence of CNS infection even though clinical signs were prevented in some groups. The greatest treatment benefits were seen in mice given 200 mg/kg ponazuril between days 4 and 14 pi. Weight gain over the course of the experiment occurred only in mice that were given 200 mg/kg ponazuril on day 7 or 10 pi. With the exception of groups given 200 mg/kg ponazuril on day 7 or 14 pi, mice in groups that got sporocysts developed abnormal neurologic signs. No deaths before day 30 pi occurred in mice given ponazuril at 20 mg/kg on day 7 pi or 200 mg/kg on day 1, 7, 10, or 14 pi. This effect was not significant. Mice given 200 mg/kg on day 7 pi had significantly fewer cerebellar schizonts than did those of the control group that was not given ponazuril. These results indicate that single-dose administration of ponazuril for prevention of CNS infection is partially protective when given between days 4 and 14 pi.

Allergen-Specific Early Neutrophil Infiltration After Allergen Challenge in a Murine Model

Allergen-Specific Early Neutrophil Infiltration After Allergen Challenge in a Murine Model

In vivo expansion of transduced murine hematopoietic cells with a selective amplifier gene.

Hematopoietic stem-cell-directed gene transfer has achieved limited success in transducing clinically relevant levels of target cells. The expansion of gene-modified cells is one way to circumvent the problem of inefficient transduction with current vectors. To this end, we have developed 'selective amplifier genes' (SAGs) that encode chimeric proteins that are a fusion of granulocyte colony-stimulating factor receptor and the steroid-binding domain. Prototype SAGs conferred estrogen-responsive growth on murine hematopoietic progenitors. We constructed a retroviral vector coexpressing an SAG for 4-hydroxytamoxifen (Tm)-specific proliferation and the enhanced green fluorescent protein (EGFP). Murine bone marrow cells were transduced with this vector and transplanted into myeloablated mice. Subsequently, recipients were challenged with Tm, and EGFP(+) cells were enumerated. The challenge induced a significant increase in EGFP(+) leukocytes (21 +/- 4% to 27 +/- 5%), while EGFP(+) cells decreased in untreated animals (21 +/- 5% to 10 +/- 3%). Three months later, bone marrow cells were transplanted from the unchallenged mice to secondary hosts. Again the administration of Tm resulted in an increase of EGFP(+) cells (16 +/- 4% to 35 +/- 3%), contrasting to a decrease in controls (22 +/- 4% to 12 +/- 4%), and the difference was significant for more than 3 months. A detailed study of lineage showed a preferential expansion of EGFP(+) cells in granulocytes and monocytes following Tm administration. Long-term repopulating cells were transduced with the SAG, and the transduced granulocyte/monocyte precursors were most likely to be expandable in vivo upon Tm stimulation.

Clearance of Helicobacter pylori Infection and Resolution of Postimmunization Gastritis in a Kinetic Study of Prophylactically Immunized Mice.

Patients infected with Helicobacter pylori mount an immune response which fails to clear the infection and may contribute to disease. Mice can be protected by immunization. To further characterize the H. pylori-mouse model, stomachs of unimmunized or intranasally immunized C57BL/6 mice were quantitatively cultured 3 days and 1, 2, 4, 8, 16, 32, and 52 weeks after challenge with H. pylori. At 3 days and 1 week after challenge, colonization was the same in the immunized and unimmunized mice. By 2 weeks after challenge, the immunized mice had a >2-log decrease in bacterial load, and at all later time points, they either were culture negative or had at least a 2-log decrease in bacterial load. Gastritis in the immunized mice peaked at 1 to 2 weeks after challenge and was characterized by a mixed inflammatory infiltrate and epithelial proliferation centered at the transition between corpus and antrum. By 52 weeks postchallenge, the gastric histology in the immunized mice was not different from that in control unchallenged mice. The unimmunized group began to show a reduction in bacterial load as early as 16 weeks after challenge, and by 52 weeks seven of eight unimmunized mice had developed gastritis and reduced bacterial loads. These results indicate that prophylactic immunization does not prevent colonization by H. pylori but enables mice to clear the infection or significantly reduce the number of colonizing bacteria. The reduction in bacterial load is associated with gastric inflammation that subsides over time.

Molecular and biological characterization of the murine leukotriene B4 receptor expressed on eosinophils.

The movement of leukocytes into tissues is regulated by the local production of chemical mediators collectively referred to as chemoattractants. Although chemoattractants constitute a diverse array of molecules, including proteins, peptides, and lipids, they all appear to signal leukocytes through a related family of seven transmembrane-spanning G protein-coupled receptors. The eosinophil is a potent proinflammatory cell that is attracted into tissues during allergic inflammation, parasitic infection, and certain malignancies. Since the molecular mechanisms controlling eosinophil recruitment are incompletely understood, we performed a degenerate polymerase chain reaction on cDNA isolated from murine eosinophils to identify novel chemoattractant receptors. We report the isolation of a cDNA that encodes a 351-amino acid glycoprotein that is 78% identical to a human gene that has been reported to be a purinoceptor (P2Y7) and a leukotriene B4 (LTB4) receptor (BLTR). Chinese hamster ovary (CHO) cells transfected with this cDNA specifically bound [3H]LTB4 with a dissociation constant of 0.6 +/- 0.1 nM. Furthermore, LTB4 induced a dose-dependent intracellular calcium flux in transfected CHO cells. In contrast, [35S]dATP did not specifically bind to these transfectants. This mRNA was expressed at high levels in interleukin 5-exposed eosinophils, elicited peritoneal macrophages and neutrophils, and to a lesser extent interferon gamma stimulated macrophages. Low levels of expression were detected in the lung, lymph node, and spleen of unchallenged mice. Western blot analysis detected the mBLTR protein in murine eosinophils and alveolar macrophages as well as human eosinophils. In addition, elevated levels of mBLTR mRNA were found in the lungs of mice in a murine model of allergic pulmonary inflammation in a time course consistent with the influx of eosinophils. Our findings indicate that this murine receptor is an LTB4 receptor that is highly expressed on activated leukocytes, including eosinophils, and may play an important role in mediating eosinophil recruitment into inflammatory foci.

Induction of a secondary human anti-HLA alloimmune response in severe combined immunodeficient mice engrafted with human lymphocytes.

Experimental manipulation of transfusion-induced alloimmunization is limited in humans by ethical considerations. Conversely, studies of alloimmunization in animal models may not reflect the human immune system closely enough to be of optimal benefit. The development of an in vivo model of human alloimmunization that is amenable to experimental manipulation is thus desirable. An in vivo model of human alloimmunization was evaluated by using mice with severe combined immunodeficiency (SCID). SCID mice underwent gamma-radiation (200 cGy) and received an intraperitoneal injection of human peripheral blood lymphocytes (PBLs) from donors immunized to HLA antigens by prior pregnancy (reconstitution). These Hu [human]-PBL-SCID mice were then challenged with HLA-mismatched PBLs. Alloantibodies were evaluated by flow cytometry and a standard two-stage microlymphocytotoxicity assay. Hu-PBL-SCID mice (n = 22) that were challenged with PBLs expressing the HLA antigens to which the donors had previously been immunized, made significantly more IgM and IgG alloantibodies than did the unchallenged mice. Responses were measurable by 1 week after reconstitution and challenge. Prior treatment of SCID mice with anti-asialo GM1, which depletes murine natural killer cells and macrophages, further increased the alloantibody response of challenged mice. The human alloantibodies generated were specific to the challenge HLA antigens as assessed by microlymphocytotoxicity assay. Hu-PBL-SCID mice are a useful model system in which to study and manipulate the induction of secondary human alloimmune responses against cellular HLA class I antigens. This model will be valuable for testing the in vivo effect of novel immunotherapies on the inhibition of the human alloantibody response.

T Cells Are Necessary for Th 2 Cytokine Production and Eosinophil Accumulation in Airways of Antigen-Challenged Allergic Mice

In a murine model of pulmonary inflammation, aerosolized antigen challenge of sensitized B6D2F1 mice leads to eosinophil accumulation within the lungs. Little is known of the role of T cells and their cytokine products in these allergic animals. In this study, we show that T cells migrate into the lungs in response to antigen challenge and are necessary for local production of cytokines (IL-4 and IL-5) important in B and T cell development as well as eosinophil activation and differentiation. Flow cytometry revealed an increase in the percentage of Thy1 + T cells but not in B220 + B cells in bronchoalveolar lavage fluid after challenge when compared to unchallenged mice. Although there was an increase in both T cell subsets, there were twice as many CD4 + cells as CD8 + cells at 24 hr and after 48 hr the CD4 + subset predominated. The CD4 + T lymphocytes were CD44 + CD45RB lo indicating an activated/memory phenotype and tracheobroncheal lymph node cells obtained from challenged mice proliferated in a dose-dependent manner in response to antigen stimulation in vitro. Reverse transcriptase-polymerase chain reaction analysis of lung tissue-derived RNA indicated an increase in Th 2-like cytokines. IL-4 and IL-5 steady-state mRNAs were at peak levels 6 hr after challenge, while no consistent increase was found for IFN-γ/mRNA levels. Treatment with the glucocorticoid betamethasone just prior to challenge reduced the levels of cytokine mRNA as well as the eosinophil influx. In vivo depletion of T cells from sensitized mice reduced pulmonary eosinophilia as well as the expression of IL-4, IL-5, and IFN-γ, steady-state mRNAs in the lungs of sensitized and challenged mice. These results indicate that T cells migrating into the lungs of mice after antigen challenge play an important role in the production of Th 2-like cytokines and the accumulation of eosinophils in bronchial fluids.

Clofibrate Pretreatment Diminishes Acetaminophen′s Selective Covalent Binding and Hepatotoxicity

Peroxisome proliferators have been shown to diminish acetaminophen (APAP) hepatotoxicity (Biochem. Pharmacol.43, 1395, 1992). To investigate the mechanistic basis for this protection CD-1 male mice were given corn oil or 500 mg clofibrate (CFB)/kg, ip, daily for 10 days. They were then fasted overnight and either killed without challenge or at 4 or 12 hr after challenge with 800 mg APAP/kg (in 50% propylene glycol). At 12 hr, hepatotoxicity was evidenced by elevated plasma sorbitol dehydrogenase and histopathology in corn oil but not in CFB-pretreated mice. At 4 hr after APAP treatment, hepatic glutathione (GSH) depletion and selective arylation of the major APAP target proteins were both greatly diminished by CFB pretreatment. Western blot analysis with the anti-58 antibody of liver cytosol from unchallenged mice showed no apparent changes in the levels of the 58-kDa major APAP target protein with CFB treatment. These findings suggest that protection could be the result of diminished net availability of generated electrophile. In vitro, measurements indicated that the specific activity in microsomes for APAP oxidation by cytochrome P450 was not changed by CFB treatment; whereas GSH S-transferase activity in cytosol was decreased by 25%. Pretreatment with CFB also produced a significant elevation in hepatic GSH. These studies indicate that protection by CFB might result from increased availability of hepatic GSH which could trap APAP electrophile nonenzymatically, thereby decreasing covalent binding and preventing toxicity.

Effect of dietary omega-3 fatty acids on cell-mediated cytotoxic activity in BALB/c mice

The effects of feeding a diet rich in omega-3 (n-3) fatty acids on immune cell prostaglandin production and cytotoxic activity before and after a viral challenge were studied in female BALB/c mice. Weanling mice were fed purified diets containing either 10% by weight corn oil (CO), 10% linseed oil (LO), or 8% menhaden oil+2% corn oil mix (FO) for 8–12 weeks. The LO diet provided 5.6 g/kg of 18:3n-3 and the FO diet 1.6 g/kg and 0.5 g/kg of 20:5n-3 and 22:6n-3, respectively. Natural killer (NK) cell activity in the unchallenged FO-fed mice was significantly lower than in CO-fed mice. Seven days after a viral challenge, cell-mediated cytotoxic (CMC) activity by splenocytes and peritoneal exudate cells from LO-fed mice was higher compared to CO and FO-fed mice. The feeding of n-3 rich diets significantly influenced the number of cells obtained from the spleen and peritoneum. Splenocyte prostaglandin E and F production was decreased by over 70% in both the LO and FO-fed, compared to CO-fed mice. The fatty acid profile of total lipids from serum and splenocytes reflected the relative n-6 and n-3 composition of the dietary fats. In summary, dietary n-3 fatty acids significantly influenced CMC activity of immune cells. Alterations in immune cell PG production do not seem to explain these differences in CMC activity. Changes in the fatty acid composition of immune cells may be an important factor.

Migration inhibition factor study in Histoplasma capsulatum

Mice sublethally infected with viable Histoplasma capsulatum or immunized with merthiolate-killed yeast phase cells showed decreased mortality on subsequent challenge infection as compared to controls. Migration inhibition (MI) assays using peritoneal and spleen cells from immunized but unchallenged mice showed no parallel correlation with percent mortality. MI assay indices fluctuated without concomitant changes in resistance to challenge injection with live yeast phase cells. Viable vaccines induced greater resistance to challenge infection than killed cells, although both were comparable in sensitizing ability as measured by MI assay techniques with this mouse model.

Growth and Invasiveness of Candida albicans in the Germ-Free and Conventional Mouse After Oral Challenge

Candida albicans was established in large numbers throughout the gut after one oral challenge in the germ-free and in the conventional mouse. Of the strains tested, only the germ-free ND 1 mouse appeared to be susceptible to infection, and this was confined to the stomach mucosa; lesions contained large numbers of hyphal and mycelial forms with blastospores. These forms were also seen in the gut of resistant germ-free ND 4 mice after challenge. Only budding yeast forms were seen in the gut contents from conventional animals. The concentration of sulfhydryl-containing compounds was decreased in the stomach contents from germ-free mice. The stomach tissue of conventional animals seemed to be more acidic than that of germ-free animals, and association of C. albicans with conventional mice neutralized some of this acidity. E h values of contents from the gut of unchallenged mice were usually higher in conventional than in germ-free animals; after challenge, the E h in both groups decreased. Some reciprocal effects of intestinal microorganisms and host are discussed in relation to intestinal candidiasis.

Growth and Invasiveness of Candida albicans in the Germ-Free and Conventional Mouse After Oral Challenge

Candida albicans was established in large numbers throughout the gut after one oral challenge in the germ-free and in the conventional mouse. Of the strains tested, only the germ-free ND 1 mouse appeared to be susceptible to infection, and this was confined to the stomach mucosa; lesions contained large numbers of hyphal and mycelial forms with blastospores. These forms were also seen in the gut of resistant germ-free ND 4 mice after challenge. Only budding yeast forms were seen in the gut contents from conventional animals. The concentration of sulfhydryl-containing compounds was decreased in the stomach contents from germ-free mice. The stomach tissue of conventional animals seemed to be more acidic than that of germ-free animals, and association of C. albicans with conventional mice neutralized some of this acidity. E(h) values of contents from the gut of unchallenged mice were usually higher in conventional than in germ-free animals; after challenge, the E(h) in both groups decreased. Some reciprocal effects of intestinal microorganisms and host are discussed in relation to intestinal candidiasis.