Gamma Knockout Mice Research Articles

Deterioration of Visual Function as Examined by Electroretinograms inToxoplasma gondii–Infected IFN-γ-Knockout Mice

To investigate by ERG the effects of Toxoplasma gondii infection on the visual function of interferon gamma knockout (GKO) mice, as a model of immunocompromised hosts. Susceptible wild-type (WT) C57BL/6 and GKO C57BL/6 mice were infected with five cysts of the avirulent T. gondii perorally. ERGs were recorded before and after the infection. The eyes of WT and GKO mice were enucleated and prepared for histologic studies 4 weeks and 12 days after infection, respectively. The a- and b-waves of ERGs did not change significantly up to 1 month after infection in WT mice, but those of GKO mice were significantly reduced 11 days after infection. Histopathology revealed focal retinitis and vasculitis in WT mice 4 weeks after infection. Mild inflammation and sludging of blood in the retina and choroid were found in GKO mice 12 days after infection, just before death. Cysts were found in the inner nuclear layer, with little disturbance of the surrounding retinal architecture in both WT and GKO mice. ERG clearly showed deterioration of visual function in GKO but not in WT mice after T. gondii infection. ERG is a sensitive and reliable method for observing activity in mice severely affected with experimental toxoplasmic retinochoroiditis.

Antitumor effect to IL-12 administration into the portal vein on murine liver metastasis.

Interleukin (IL)-12 has been shown to possess potent antitumor activity, and an antitumor effect of systemic IL-12 administration has been reported in liver metastasis models. In this study, we examined the usefulness of local IL-12 administration into the portal system in the treatment of liver metastasis. First, we confirmed the antitumor effect of recombinant IL-12 (rIL-12) on MC-38 tumors in an intracutaneous model. In the murine liver metastasis model, 1 x 10(5) of MC-38 cells were injected into the portal vein on day 0, and the spleen was transpositioned subcutaneously for administration of rIL-12 continually into the portal system. From days 3 to 7, 0.1 micro g rIL-12 was administered intraperitoneally or intrasplenicly, while Hanks' Balanced Salf Solution (HBSS) was injected intrasplenicly in the control group. The liver weight in the rIL-12 intraperitoneal treatment group (1.88 +/- 0.37 g) and that in the rIL-12 intrasplenic treatment group (1.43 +/- 0.21 g) were significantly less than that in the HBSS group (2.86 +/- 0.74 g; P < 0.05). The numbers of metastatic nodules in the rIL-12 intraperitoneal treatment group (22.3 +/- 17.1) and in the rIL-12 intrasplenic treatment group (12.4 +/- 13.8) were significantly less than that in the HBSS group (137.1 +/- 44.9; P < 0.05). Complete regression of the tumor was observed in one of six mice in the rIL-12 intrasplenic treatment group. This antitumor effect of rIL-12 on MC-38 liver metastasis was not observed in interferon (IFN)-gamma knockout mice. Intraportal administration of IL-12-transduced fibroblasts, which were syngeneic to C57BL/6 mice, had an antitumor effect in the MC-38 liver metastasis model. These results suggested that the local administration of IL-12 into the portal system would be a useful strategy for the treatment of liver metastasis.

Salmonella typhi and S. typhimurium derivatives harbouring deletions in aromatic biosynthesis and Salmonella Pathogenicity Island-2 (SPI-2) genes as vaccines and vectors

The S. typhimurium strain (TML Δ aroC Δ ssaV) WT05, harbouring defined deletions in genes involved in both the aromatic biosynthesis pathway ( aroC) and the Salmonella Pathogenicity Island-2 (SPI-2) ( ssaV) was shown to be significantly attenuated in C57 BL/6 interferon gamma knockout mice following oral inoculation. Similarly, the S. typhi strain (Ty2 Δ aroC Δ ssaV) ZH9 harbouring the aroC and ssaV mutations propagated less efficiently than wild type in human macrophages. These studies demonstrated the attractive safety profile of the aroC ssaV mutant combination. Strains S. typhimurium (TML Δ aroC Δ ssaV ) WT05 and S. typhi (Ty2 Δ aroC Δ ssaV) ZH9 were subsequently tested as vaccine vectors to deliver E. coli heat-labile toxin (LT-B) mucosally to mice. Mice inoculated orally with S. typhimurium (TML Δ aroC Δ ssaV) WT05 expressing LT-B (WT05/LT-B) elicited high titres of both LT-specific serum IgG and intestinal IgA, although no specific IgA was detected in the vagina. Similarly, intranasal inoculation of mice with S. typhi (Ty2 Δ aroC Δ ssaV) ZH9 expressing LT-B (ZH9/LT-B) elicited even higher titres of LT-specific serum antibody as well as LT-specific Ig in the vagina. We conclude that Δ aroC Δ ssaV strains of Salmonella are highly attenuated and are promising candidates both as human typhoid vaccines and as vaccine vectors for the delivery of heterologous antigens.

PPARgamma, an X-ceptor for Xs.

Evidence from both human genetic studies and characterization of peroxisome proliferator-activated receptor gamma (PPARgamma) knockout mice suggested that the prime function of PPARgamma is fat formation and that its role in insulin sensitization might be secondary to this function. The thrifty function of PPARgamma was most likely evolutionary beneficial, but might in "times of plenty" contribute to the pathogenesis of disorders, such as obesity, insulin resistance, type 2 diabetes, and hyperlipidemia, often commonly referred to as "syndrome X". This role of PPARgamma in these diseases also questions the eventual therapeutic benefits of pure PPARgamma activation, which is associated with an increase in adipose tissue mass. We characterized a new chemical class of PPARgamma agonists, that is, FMOC-l-leucine (FLL). FLL induces a different conformation of PPARgamma relative to classical PPARgamma ligands. Mass spectrometry indicates that two molecules of FLL bind to a single PPARgamma molecule, making its mode of receptor interaction distinctive. FLL recruits a different set of coactivators and activates PPARgamma with a lower potency, but a similar maximal efficacy, relative to known PPARgamma ligands. In contrast, FLL is a more effective insulin sensitizer than current PPARgamma agonists, an effect potentially linked to its weak adipogenic activity. These data make a strong point for potential therapeutic benefits of PPARgamma modulation rather than activation.

Phenotypic analysis of oral tolerance to alloantigens: evidence that the indirect pathway of antigen presentation is involved.

Oral administration of alloantigens induces down-regulation of Th1 immune responses and reduces the incidence of corneal graft rejection. This study examined the role of Th1 and Th2 cytokines, accessory cells, and lymphoid organs that are known to be instrumental in other forms of antigen-specific tolerance. Allogeneic dendritic cells (DC) were administered orally using a protocol that is known to reduce the incidence of corneal allograft rejection and prevent the generation of allospecific delayed-type hypersensitivity (DTH). Hosts included normal mice and gene knockout (KO) mice, including B cell-deficient (mu)MT, interleukin (IL)-4 KO, IL-10 KO, and interferon (IFN)-gamma KO mice. The requirement for either an intact spleen or thymus was also examined. Orally administered paraformaldehyde-fixed, UVB-treated, or sonicated allogeneic cells were tested to determine if dead cells were capable of inducing tolerance. Studies on gene KO mice indicated that a Th1 cytokine (IFN-gamma) and a Th2 cytokine (IL-4) were needed for the development of oral tolerance to alloantigens. By contrast, IL-10 was not required. Although an intact spleen was necessary for the development of tolerance, removal of the thymus did not affect down-regulation of DTH. Oral tolerance induced with allogeneic cells shares characteristics with antigen-specific unresponsiveness induced by other routes, yet there are some noteworthy differences. The capacity of killed or sonicated allogeneic cells to induce oral tolerance and enhance corneal graft survival indicates that oral tolerance to alloantigens can occur via the indirect pathway of alloantigen presentation. These results also emphasize the remarkable redundancy in the mechanisms that the immune system employs to produce antigen-specific unresponsiveness.

Interleukin-18 regulates acute graft-versus-host disease by enhancing Fas-mediated donor T cell apoptosis.

Interleukin (IL)-18 is a recently discovered cytokine that modulates both T helper type 1 (Th1) and Th2 responses. IL-18 is elevated during acute graft-versus-host disease (GVHD). We investigated the role of IL-18 in this disorder using a well characterized murine bone marrow transplantation (BMT) model (B6 --> B6D2F1). Surprisingly, blockade of IL-18 accelerated acute GVHD-related mortality. In contrast, administration of IL-18 reduced serum tumor necrosis factor (TNF)-alpha and lipopolysaccharide (LPS) levels, decreased intestinal histopathology, and resulted in significantly improved survival (75 vs. 15%, P < 0.001). Administration of IL-18 attenuated early donor T cell expansion and was associated with increased Fas expression and greater apoptosis of donor T cells. The administration of IL-18 no longer protected BMT recipients from GVHD when Fas deficient (lpr) mice were used as donors. IL-18 also lost its ability to protect against acute GVHD when interferon (IFN)-gamma knockout mice were used as donors. Together, these results demonstrate that IL-18 regulates acute GVHD by inducing enhanced Fas-mediated apoptosis of donor T cells early after BMT, and donor IFN-gamma is critical for this protective effect.

Migration and development of Sarcocystis neurona in tissues of interferon gamma knockout mice fed sporocysts from a naturally infected opossum

Migration and development of Sarcocystis neurona was studied in 50 gamma interferon knockout mice fed graded doses of S. neurona sporocysts from the intestine of a naturally infected opossum. Mice were examined at necropsy 1–62 days after feeding sporocysts (DAFS). All tissue sections were reacted with anti- S. neurona-specific polyclonal rabbit serum in an immunohistochemical (IHC) test. Between 1 and 3 DAFS, organisms were seen mainly in intestines. Between 4 and 11 DAFS, organisms were seen in several visceral tissues. Beginning with 13 DAFS, schizonts and merozoites were present in sections of brains of all infected mice. All regions of the brain were parasitized but the hind brain was most severely affected. S. neurona was found in the spinal cord of all 10 mice examined 22–30 DAFS. Of the 28 infected mice examined 20–62 DAFS, S. neurona was found in the brains of all 28, lungs of 14, hearts of 8 and eyes of 3. More organisms were seen in IHC-stained sections than in sections stained with hematoxylin and eosin. Treatment of tissues with glutaraldehyde, Karnovsky fixative, and ethylene diamino tetra acetic acid (EDTA, used for decalcification) did not affect staining of organisms by IHC.

Optimal conditions for establishment of experimental tuberculosis model using an automated inhalation exposure apparatus and its application

Animal (mouse and guinea pig) pulmonary tuberculosis models were established, using an automated inhalation exposure apparatus (Glas-Col Corp., USA, Model 099CA-4212). This apparatus includes four steps--preheating, nebulization, cloud decay and decontamination. The optimal conditions for M. tuberculosis H37Rv strain infection experiments were as follows: 10(5-6) colony forming unit (cfu) tubercle bacilli; preheating for 15 min.; nebulization for 90 min.; cloud decay for 15 min. and decontamination for 5 min. When 10(4) cfu M. tuberculosis H37Rv strain were introduced into the lungs of interferon (IFN)-gamma knockout mice, using the inhalation exposure apparatus and were followed up for 9 months, the primitive cavitary lesions were observed. This apparatus was also useful for inhalation exposure experiments of guinea pigs. This apparatus can also be utilized for animal inhalation experiments of allergens.

Interleukin-10 gene transfer to the airway regulates allergic mucosal sensitization in mice.

The objective of this study was to investigate the effect of airway gene transfer of interleukin (IL)-10, a cytokine with potent anti-inflammatory and immunoregulatory activities, on allergic mucosal sensitization. We used a recently described murine model that involves repeated exposures to aerosolized ovalbumin (OVA), daily for 10 d, in the context of granulocyte macrophage colony-stimulating factor (GM-CSF) expression in the airway environment achieved by intranasal delivery of a replication-deficient adenovirus carrying the GM-CSF transgene. The resulting inflammatory response was characterized by a T-helper 2 cytokine profile and marked airway eosinophilia. After complete resolution of the inflammatory response (Day 28), a single exposure to OVA reconstituted airway eosinophilia and induced airway hyperresponsiveness. We show that concurrent expression of IL-10 inhibited GM-CSF-driven OVA-specific inflammation in a dose-dependent manner. Specifically, IL-10 decreased the number of mononuclear cells, neutrophils, and eosinophils in the bronchoalveolar lavage fluid (BALF). Histologic evaluation of the tissue corroborated the findings in the BALF. Concurrent expression of IL-10 at the time of mucosal sensitization abrogated both the cellular and physiologic recall responses in vivo. Studies in interferon (IFN)-gamma knockout mice demonstrated that prevention of airway eosinophilia by IL-10 was IFN-gamma-independent and that expression of IL-10 was associated with decreased levels of IL-4, IL-5, and tumor necrosis factor-alpha in the BALF. Flow cytometric analysis of dispersed lung cells showed that expression of IL-10 in the airway reduced the absolute number of Class II major histocompatibility complex (MHC)(+)/CD11c(+) (dendritic cells) and Class II MHC(+)/Mac-1(bright) (macrophages) cells expressing the costimulatory molecules B7.1 and B7.2 by 30%. However, IL-10 coexpression did not prevent expansion of CD4 and CD8 T cells or expression of the early activation marker CD69 on T cells. Thus, airway gene transfer of IL-10 altered the immune response to OVA in a way that resulted in inhibition of airway inflammation. These findings suggest that development of an immunoregulatory strategy based on IL-10, alone or in combination with GM-CSF, warrants further consideration.

Anti-immunoglobulin E treatment decreases worm burden and egg production in Schistosoma mansoni-infected normal and interferon gamma knockout mice.

The immunoglobulin E (IgE) response is generally considered an essential component of the host defense against parasitic helminths such as Schistosoma mansoni. In contrast, work on antischistosome vaccines suggests that interferon gamma (IFN-gamma) is the critical immune mediator for vaccine-induced immunity to the parasite. In this study, the total IgE response to a primary S. mansoni infection was suppressed by anti-IgE treatment in both normal mice and in mice with defective IFN genes (gene knockout [GKO]). Reduction of the IgE response resulted in decreased worm burden and a decrease in the number of eggs produced per worm in both normal and GKO mice. Whereas anti-IgE treatment also resulted in reduced hepatosplenomegaly, granulomas around existing schistosome eggs showed normal cellularity. Serum interleukin 4 levels fell in response to the reduction in serum IgE as well. The data suggest that IgE plays a detrimental, rather than beneficial, role for the host in schistosomiasis. Furthermore, the absence of IFN-gamma was found to be of little consequence to the host-response to adults or eggs in a primary schistosome infection.